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STANDARDIZING EMBRYO RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE NEW VARIABILITY ABSTRACT THESIS SUBMITTED FOR THE DEGREE OF Boctor of $i|tlo£io9i)P M BOTANY GHAZALA PRAVEEN DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA) 2002 r«. « ( «! (•

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Page 1: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

STANDARDIZING EMBRYO RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE NEW VARIABILITY

ABSTRACT

THESIS SUBMITTED FOR THE DEGREE OF

Boctor of $i|tlo£io9i)P M

BOTANY

GHAZALA PRAVEEN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA)

2002

r « .

« ( «! (•

Page 2: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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0 9 JUM 2035

Page 3: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

India has been a major importer of wheat grain till 1960s. It was

with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64

and Sharbati Sonora, Kalyansona, Sonalika, Arjun, WL 711, HD 2329,

PBW 343 etc, the green revolution resulted increasing wheat production

to the extent that India became 2"* largest wheat producing country in the

world in 1999. One of the major drawbacks of the green revolution is that

genetic erosion of the indigenous genotypes/native germplasm pool

occurs due to dependence on few selected types only and thus making the

crop more vulnerable to various diseases and pests. The new varieties

released every year do not last long as new pathotypes appear and also

old resistant genes get defeated due to development of virulence against

the existing ones. Since success of any crop improvement program very

much depends on the extent of genetic variability present in the base

population, it is required to broaden the genetic base by enriching the

crop with more and more desirable genes.

Wild relatives of wheat are a valuable reservoir of genes which are either

absent in cultivated wheat or their diversity is narrow particularly with respect to

pest resistance, tolerance to biotic and abiotic stresses and various morphological

traits including the end use properties for making bread and pasta as well as other

products. Therefore, the wild species can be used as a potential source to add

novel variability and affecting the genetic enrichment program through wide

crossing. However, in most of the wide crosses the embryo aborts at various

levels of development, Hence, the technique of embryo rescue involving excision

and culture of embryos aseptically had been quite successful in transferring the

useful traits from alien sources to the background of cultivated species.

During the present course of study, before going straight for rescuing

embryo, an attempt has been made to develop a suitable regeneration protocol

Page 4: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Ahslracl 2

and to select out the better responding genotype in vitro. Although, several

other factors can affect the callus induction and plant regeneration from

rescued embryo. Besides, an established protocol can also help in going for

transformational studies through various methods, inducing somaclonal

variation, screening of genotypes against various stresses and in vitro

mutational studies.

In lieu of this, an effort was made to standardize a regeneration protocol,

using the cultivars of both 71 durum and T. aestivum species of wheat. Later on,

the embryos of the wide crosses were rescued to test the protocol on selected

varieties and media.

The salient results/observations recorded from the present study have

been summarized here under:

> Two varieties each for bread wheat (WH 542 and UP 2338) and durum

wheat (Bijaga Yellow and Gulab) were screened on four callus induction

media CI (MS+2.4-D, 2mg/l), C2 (MS + 2.4-D, 4mg/l), C3 (MS+ 2,4-D,

2mg/l + lAA. 0.5 mg/1) and C4 (B5 + 2,4-D, 2mg/l). The embryos were

excised at 10, 12, 14, 16 and 18 days post pollination/days post anthesis.

12 and 14 days calli of bread wheat and 12, 14 and 16 days old calli of

durum wheat were found optimum. Maximum frequency of callus

induction was recorded on C1 (embryogenic) than on C2 (loose and non-

embryogenic)

> Five media viz., Rl (left on the same medium and exposing to light), R2

(MS + Kinetin, 2 mg/1), R3 (MS + Kinetin, 2mg/l + lAA, 0.5 mg/1), R4

(MS + BAP, 2 mg/1) and R5 (MS +BAP, 2ml/l + lAA, 2mg/l) were tested

to achieve regeneration from calli produced on CI, C2, C3 and C4 media.

> Three media viz., Rl, R2 and R5 were found to give good regeneration in

all the varieties, while R3 and R4 proved quite poor in this regard.

Page 5: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Abstract 3

> The regenerated plantlets were grown on three media viz., SI (left on the

same medium where they were induced), S2 (MS + GA3 0.5 mg/1 +30 g/1

sucrose) and S3 (GR free MS + 20 g/1 sucrose) up to 3 to 4 leaf stage.

Among these, the S3 medium was found better than other two media.

> The regenerated plantlets were transferred on a range of rooting media

and the medium containing MS salts without vitamins + NAAimg/1 +

sucrose 20g/l) was found better responding than rest.

> The dark condition during callusing and light during regeneration phase

proved quite effective. Interestingly, the alternate light and dark

treatment using 20 watts electric bulbs (16/8 hrs light/dark) could induce

regeneration in calli produced on MS + 2, 4-D (2mg/l) medium.

> A temperature of 25±2°C was found optimum, while the further rise in

temperature resulted in the browning of callus.

> The plantlets were hardened in small pots filled with agropeat and soilrite

in equal proportions and put into growth chamber at 15±1°C and 90 per

cent relative humidity. Established plants were transferred in bigger pots

containing soil, sand, FYM and agropeat in equal proportions.

> Following the above mentioned protocol, six additional varieties each of

bread wheat (CPAN 3004. KRL 1-4. PBW 343, PBW 373. Raj 3765 and

HD 2329) and durum wheat (HD 4530. HI 8381, Raj 1555. PDW 215.

PDW 233 and MACS 2846) were screened to check both the validity of

protocol and selection of better responding genotypes.

> The age of immature embryo, genotype, media composition and culture

conditions all affected the regeneration potential of the explant.

Page 6: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Abstract 4

> The above mentioned protocol was further extended to grow hybrid plants

from the crosses involving wide relatives.

> Five varieities each of bread wheat (WH 542, UP 2338, HD 2329, PBW

343 and Raj 3765) and durum wheat (Bijaga Yellow, Raj 1555. Gulab,

PDW 215 and PDW 233) were taken as female and 20 species of wild

triticum and Aegilops species [T. urartu (A"), T. hoeticum (A**), Ae.

speltoides(S), Ae. longissima (S), Ae. sharonensLs{S^^^), Ae. hicornis (S^),

Ae. squarrosa (D), Ae. caudata (C), Ae. markgrafii (C), Ae. umbellulata

(U), Ae. cylindrica (CD), Ae. ventricosa (D"), Ae. kolschyi (US), Ae.

peregrine (US), Ae. triuncialis (UC), Ae. ovata (UM"), Ae. lorentii

i\JM\ Ae. triaristata (UM'), Ae. geniculata{\JM°), T. araraticufn(AG)]

were used as pollen parent.

> The hand emasculated spikes were pollinated twice with pollens collected

from the alien species. The pollinated spikes were sprayed with 2. 4-D +

GA3 solution for four consecutive days starting from the day of

emasculation.

> The hybrid embryos were scooped out at 10, 12, 14 and 16 days with

slight variation in some crosses and cultured aseptically following the

above mentioned protocol.

> The embryos rescued after 12 to 14 DPP were found to give optimum

regeneration in most of the crosses.

> The regenerated hybrid plants were established in the field with various

levels of seed settings.

Page 7: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

\

STANDARDIZING EMBRYO RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE NEW VARIABILITY

THESIS SUBMITTED FOR THE DEGREE OF

Boctor of $))tIo£iop{)p M

BOTANY VV

W

- > \

< ^ . '

GHAZALA PRAVEEN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202 002 (INDIA)

2002

Page 8: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

/

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T5841

Page 9: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

PLANT TISSUE CULTURE & CYTOGENETICS LABORATORIES

Department of Botany, Aligarh Muslim University, Aligarh.202 002 (INDIA)

CERTIFICATE

Certified that the thesis entitled "STANDARDIZING EMBRYO

RESCUE TECHNIQUE IN TRITICUM SYSTEM TO GENERATE

NEW VARIABILITY " embodies the original research work carried out by

Ms. GHAZALA PRAVEEN under our guidance and supervision. The

work has not been submitted elsewhere for the award of any other degree or

diploma and can be submitted in fulfillment of the requirement for the

award of the degree of Doctor of Philosophy in Botany of AUgarh Muslim

University, Aligarh.

Dr. Mohammad Anis Supervisor Plant Cytogenetics and Tissue Culture Lab Department of Botany, Aligarh Muslim University AUgarh 202 002 (India)

y^^r^y^

Dr. Jag Shoran Co-Supervisor

Principal Investigator Crop Improvement Division

Directorate of Wheat Research (Indian Council of Agricultural Research)

Kamal-132 001 (India)

Page 10: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

VedlccubeJi

to

Page 11: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Ii4i^e6sezC by tke^ dUcoutrL&i of gireaor Mt*uLei, BlffeMy

LH/ 1906 i^^OAiateA'.

"tf t Huw Loolc into tke^juiwre^ t aiH/jhiresee-

the' tmte' COIMIHO wke*v « ^ skaH be^ oJbLe^ to

UKLte^ uv onZ' tmriety the best atiaiUy, the best

cropping poci^tr, the best strcuit MUL SO o^t

After alt4wst cu ctntuyy oftUajit brttdlna n/e^ are^ still uv the

Huddle but the iMiMMlnatioH/ of com^eteMt ploMt breeders

like B(£fzH/ stiMudatts lu to specMiatefurther:

Page 12: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

AC1CN'0M/L<E(Dg^M<E!Nr

To Begin xintfi I Bow in reverence to the JiCmigfity, as it is indeed!Kis BCessings,

wfiicfi (edrm to complete this piece ofworf{^

It is my proud privilege to ej(press my deep sense of gratitude and sincere thanf{s to

my supervisor (Dr. MohammadJinis, ^aderin the (Department of (Botany, JiCigarhMusCim

University, Jidgarh and my co-supervisor (Dr. Jag Shoran, (PrincipaC, Scientist, (Directorate

of 'Wheat (Research, 'Karrw.C for Benevolent guidance, constant watchfuC interest and

heCping attitude throughout the present investigation.

I am ex}reme[y thankful to (Dr. S. !Kagarajan, 'E^Q^oject (Director, (Directorate of

'Wheat (Research for alfoiving me to carry out my ej^periment at the (Directorate of'Wheat

(Research, T(amaC My thanlifuCruiss is also ej(tended to (Prof Saeed^. Siddiqui, Tji-

chairman, (Department of (Botany and (Prof SamiuC[ah, Chairman, (Department of (Botany

Jlligarh MusCim 1)niversity, JiCigarhfor mailing things convenient for me to Cay down my

eo(;periment at T^amaC

It is my great pleasure to record my deep sense of gratitude and irufeBtedness to (Dr.

(B.S. Tyagi, Senior Scientist, for his ej(ceClent guidance, meticuCous supervison and healthy

criticism throughout the present investigation and preparation of manuscript, vAthout

which it would have not Been possiBle to Bring the thesis in the present form.

I ackriowCedge (Dr. (D.S. Chauhan, (P.I.-Agronomy, (Dr. (5.%, Misra,

(P.I.-QuaCity and (Basic Sciences, ©r. S. Sharma, (P.I.-(P[ant (Protection and

(Dr. 9^. Singh, (P. I.-Computers and Statistics for extending their heCp whenever required.

I also express my sincere thanl{s and gratitude to (Dr. (Raj "Kumar for providing

faciCities aruf infrastructure for the experimental wor^(Dr. (B.S- MaCi^ requires a special

mention for procuring necessary manpower

The moral support from (Dr. (Rjitan Tiwari, (Dr. < 7C ^upta, SeniorScientists and

my friends ^agan (Bajwa, Moushami (Dutt aruffatima M. Sultana proved to Be afource

of Inspiration and zest for me.

Page 13: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

/ am very mucfi tfianfiJuCto (Dr. Sfiafiid ^hmacC and Mr. (Rajendra % goCafor

their support, immense care, irritating criticism (?) and unconditionaC fieCp. Jissistance

extended to me 6yMr. T^eeraj Tyagi, Mr. Harsh Qhoudhary, Mr. <Pradeep %umar, (Dr.

AnwarShahzad, Mr. Mohd<Faisa(, Mr. M. KashifJfusain, Mr. 9/aseemJLhmadisdu(y

acf^nowtedge. Co-operation of Mr. "Kjizi 'Fariduddin and (Dr. Jlrif "Wad is also

acfinowCedged.

Services extended 6y Mr. ([(fljendra Sharma, (Photographer and Mr. yiagar,

Librarian needs a speciaC mention. The personal attention given By Mr. JlniCforthe entire

computerjoS related to my thesis is commendaBle.

I also stand oSCiged to aU those people -who woriied Behind the curtain with great

sincerity and utmost seCfkssness to maf{e this project a success and to whom I cannot

ej(press individuaCgratitude.

(Research associateship provided By the (Department of(BiotechnoCogy, !New (DeChi

through (Directorate of'Wheat (Research, 'KiamaCis duly acfijiowledged.

(Finally, no words can match the emotionaC succor and amorous Cove which I got

from my (Parents, sistersShaBana, Sameena,Arzoo, SheeBa, BrotherAamirandMu^rram

JIfi during the entire course of my study. It was that intangiBle support mechanism which

helped me in proving my self as a denoted scholar and worthy memBer of concerned

fraternity.

f ( X ^ ^ ^

(ghazaCa (Praveen)

Page 14: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Contents Chapter Title Page No

1. Introduction

1.1 Wheat crop improvement, genetic variability and alien gene transfer

1.2 Wide crossing: history and prospects

1.3 Wild Triticum and goat grass : a useful reservoir

1.4 Constraints to wide crossing

1.6 Study undertaken

2. Review of Literature

2.1. Embryo culture

2.2. Wide hybridization

2.3. Constraints to wide hybridization

2.4. Stratagies to exploit alien sources

2.4.1. Choice of promising and compatible parents

2.4.2. Crossing procedure and pre/post pollination treatments

2.5. Embryo rescue

2.5.1. Immature embryo culture

2.5.1.1. Stage of scooping out

2.5.1.2. Culture pathways

2.5.1.3. Media manipulation

2.5.6. Alternative methods

2.6. Alien genes transferred in wheat

3. Materials and Methods

3.1. Procurement of wheat germplasm

3.2. Alien germplasm

3.3. Harvesting of immature embryos and wide crossing

3.4. /« v/Yro culture

3.4.1. Basal culture medium

3.4.1.1 Immature embryo culture

3.4.1.2. Rescued embryo culture

3.4.2. Growth regulators

3.4.3. Preparation of culture medium

1-9

4

6

7

9

10-22

11

16

16

17

17

18

19

19

19

20

20

21

22

23-30

23

24

25

26

26

26

26

26

26

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Chapter

3.5.

3.6.

3.7.

3.8.

4. Results

4.1.

4.2.

4.3.

Asepti

Title

ic technique

Sterilization and plating of plant material

Incubation of cultures

Hardening of the plantlets

Standardization of regeneration protocol

4.1.1.

4.1.2.

4.1.3.

4.1.4.

4.1.5.

4.1.6.

Callus induction

4.1.1.1. Effect of callus induction media

4.1.1.2. Influence of age of embryos (DPP)

Regeneration

4.1.2.1. Bread wheat

4.1.2.2. Durum wheat

Interaction of callus initiation and regeneration medium

4.1.3.1. Bread wheat

4.1.3.2. Durum wheat

4.1.3.3. Statistical inference

Shoot proliferation and plantlet elongation

4.1.4.1. Bread wheat

4.1.4.2. Durum wheat

4.1.4.3. Statistical inference

Rhizogenesis in regenerants

4.1.5.1. Inference

Hardening and acclimatization

Selection of the better responding genotypes

4.2.1.

4.2.2.

4.2.3.

Callusing

Precocious germination

Regeneration

4.2.3.1. Regeneration on R1 medium

4.2.3.2. Regeneration on R2 medium

4.2.3.3. Regeneration on R5 medium

4.2.3.4. Selection of the varieties

Embryo rescue and wide crossing

Page No

28

28

29

29

31-61

31

31

31 T "1

34

34

37

37

39

39

39

39

41

41

41

43

43

45

45

45

47

47

47

47

50

53

Page 16: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Chapter Title Page No

5. Discussion 62-71

5.1. Basal culture media 62

5.2. Growth regulator 63

5.3. Precocious germination 64

5.4. Age of embryos 65

5.5. Growth condition of cultures 66

5.6. Genotype 67

5.7. Wide crossing and embryo rescue 68

6. Summary and Conclusion 72-74

Bibliography i-xxi

Annexure I-III

Page 17: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

List of Tables

No. Title Page No.

Table 1: Alien wheat sources for specific traits 6

Table 2: Factors influencing in vitro responses of immature 12 embryos of wheat

Table 3: Disease resistance genes incorporated in wheat gene 22 pool from alien sources.

Table 4: Varieties used in standardization of regeneration 23 protocol from immature embryos and selection of genotypes to be used in wide crossing.

Table 5: Varieties selected for wide crossing program 24

Table 6: Wild species used in crossing program 24

Table 7: Recipe of different MS medium's used (final volume 1 27 liter).

Table 8: Effect of age of embryos (days post pollination, DPP) 32 and media composition on callus induction and precocious germination (data recorded 25 days after culture).

Table 9: Percent regeneration on different media in bread wheat 3 5 varieties (data recorded 25 days Qftet tuLtuYe).

Table 10: Percent regeneration on different medium from durum 36 wheat varieties (data recorded 25 days after culture)

Table 11: Influence of media composition and age of embryos 3 8 (days post pollination, DPP) on effective regeneration in bread wheat.

Table 12: Influence of media composition and age of embryos 40 (days post pollination, DPP) on effective regeneration i.e. regeneration per 10 explant cultured in durum wheat.

Table 13: Number of shoots (having at least 3 nodes) per callus 42 and per 10 ex-plant (data recorded 20 days after sub culture)-

Table 14: Rooting efficiency of in vitro regenerated shoots of 44 bread and durum wheat cultivars •

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No. Title Page No.

Table 15: Per cent callus induction and precocious germination 46 from the immature embryo ex-plant of bread and durum wheats on MS+2,4D (2mg/l) over different days post anthesis (dpa)

Table 16: Per cent regeneration on Rl medium from calli 48 induced on CI (data recorded 25 days after transferring on to regeneration medium)

Table 17: Per cent regeneration on R2 medium from calli 49 induced on CI (data recorded 20 days after culture)

Table 18: Per cent regeneration on R5 medium from calli 51 induced on CI (data recorded 25 days after transferring on regeneration medium)

Table 19: Comparative chart for callusing and regeneration 52 potential of some bread and durum wheat varieties

Table 20: Wide crosses performed to standardize embryo rescue 54 technique

Table 21: Differential responses to callus induction and 57 regeneration of rescued embryos of different age group

Table 22: Cross combinations where dissectible embryos could 61 not be obtained

Table 23: Rescued embryos showing no response to callus 61 induction

Page 19: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

List of Figures

Fig. 1: Genetic gain in yield after insertion of alien genes

Fig. 2: Possible strategy for exploitation of genetic resources and advanced genetic

technique in a long term breeding program.

Fig.3: Different types of induced calli

Fig.4: Morphogensis in different types of calli

Fig. 5: Different development stages

Fig.6: Hardening and acclimatization of plantlets

Fig.7: Normal and abnormal embryos rescued from wide crosses

Fig. 8: Regeneration from embryos along with peculiar hybrid spikes.

Fig. 9: T. urartu crosses at different stages

Fig. 10: Different wild hybrid plants established in field through embryo rescue

Page 20: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

ZNTKOPUCTtON

Page 21: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

1. Introduction

Wheat is an important and most ancient cereal crop in the world. The

variability present in the cultivated and wild relatives of wheat, traces its

origin to the evolution of angiosperms. Over the period, the wheat has

undergone various divergent and convergent evolutions leading to the modern

polyploid form. It belongs to the genus Tritlcum that comprises around 500

species. The commonly cultivated species are bread and durum wheats, the

dicoccum being cultivated only in certain pockets including the peninsular

part of India. Wheat is cultivated in one or the other part of the globe

throughout the year. In India aestivum, durum and dicoccum wheat cultivation

is in practice. T. aestivum acquires prime position with more than 90% share,

while T. durum, the second major species, contributes around 4% of total

wheat production.

Wheat is a unique gift of nature to mankind as it can be molded into

innumerable products like chapaties, bread, cakes, crackers, biscuits, pastries,

pizzas, patties, pasta, noodles, macaroni, sphagetties etc. Wheat is consumed

by nearly 35% of world population and contributes 20% food calories. In

India the availability of wheat has increased from about 79g to more than

185g/head/day despite doubling the -. ^ population since 1961. The

increase in protein availability due to reduced availability of pulses from

69g/head/day in 1961 to dihoxxi 38g/head/day has been compensated by the

increased availability of wheat. It is also reported to contribute more calories

and protein to the world's diet than any other cereal crop (Hanson et al.,

1982), sharing about 40% of total cereal protein production. The major

protein fractions are albumin, globulin, protease, gliadin and glutelin.

Moreover, it is also good source of vitamins of the B complex group and E.

The common bread wheat (6x) and durum wheat (4x) both are true

breeding natural hybrids. Bread wheat is an allohexaploid (2n=6x=42),

Page 22: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Inlrocluclion 2

composed of three genomes A, B and D. derived from three diploid wild

species native to the Middle East. The donor of A genome is Triticum urartu

Turn. The source of B genome is Aegilops speltoides Tousch. The D genome

came from Aegilops squarossa L. [= Ae. tauschii Coss, T. tauschii (Coss.)

Schmalti]. The cytoplasm of wheat is known from B genome donor. Durum

wheat is the fore runner of bread wheat, an allotetraploid (2n=4x=28) with

two genomes A and B. It is a result of natural hybridization between T. urartu

and Ae. speltoides. The hexaploid wheat evolved latter via natural

hybridization of durum wheat with a third wild grass Ae. squarrosa which

contributed its D genome (Jauhar, 199 ) . This extraordinary series of

hybridization produced both the durum and the bread wheat that sustain

mankind.

India used to be a major importer of wheat grain till 1960s. It was with

the advent of semi dwarf wheat varieties like Lerma Rojo, Sonara 64,Sharbati

Sonora, Kalyansona, Sonalika, Arjun, WL 711, HD 2329, PBW 343 etc. a

green revolution set in increasing wheat production to the extent that India

become 2"'' largest wheat producing country in the world in 1999. Now, it is

the time to think about sustaining this increase in yield per se and improving

the grain quality for export purpose. One of the major drawbacks of the green

revolution is that genetic erosion of the indigenous genotypes/native

germplasm pool occurs due to dependence on few selected types only and thus

making the crop more vulnerable to various diseases and pests. The new

varieties released every year do not last long as new pathotypes appear and

also old resistant genes get defeated due to development of virulence against

the existing ones. Therefore, there is always an urge to incorporate new genes

for different biotic and abiotic stresses.

Since success of any crop improvement program very much depends on

the extent of the genetic variability present in the base population, it is

required to broaden the genetic base by enriching the crop with more and

more desirable genes. Even long term breeding objectives can not be realized

unless more genetic variability is generated. Further, several international laws

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Introduction J

like Intellectual Property Rights (IRP). General Agreement on Trade and

Tariffs (GATT), Plant Variety Rights (PVR), etc. are either been framed/or

likely to be framed which may restrict the inflow of germplasm being

provided by other countries. Hence, national program should be self-sufficient

and least dependent on the external sources.

1.1 WHEAT CROP IMPROVEMENT, GENETIC VARIABILITY AND ALIEN GENE TRANSFER

With the development and adoption of high yielding wheat varieties

with narrow genetic base over large growing areas, wheat crop has become

more vulnerable to biotic and abiotic stresses. Also, tremendous variability

existing in the indigenous landraces and wild germplam has been eroded and

facing extinction. As a result, the plant breeders find less and less appropriate

germplasm with desired traits among cultivated crops. The eroding genetic

base of the cultivated wheat has lead the research workers to investigate the

possibility of utilizing the genetic variation present in the wild relatives

(Feldman and Sears. 1981).

At present, there is lack of genes for some of the important characters

to be used in wheat breeding. Particularly, it is true for resistance to wheat

diseases that are either absent in the common wheat genebank or their

diversity is insufficient to support the wheat breeding program.

There are a number of ways to create and add new variability in

the wheat germplasm. The wild crosses are one such opportunity available

with the wheat geneticists. The secondary gene pool of wiJd wheat and alien

species of Aegilops, Agropyron, Elymus and other related genera that have

been reported to constitute a rich repository of resistance to various diseases,

stresses, quality characters etc. and can effectively contribute to genetic

enrichment program (Dhaliwal and Gill 1982; Gill et al., 1983; Tomerlin et

al., 1984; Mosemen et al., 1985; Warham et al., 1986; Jauhar 1993, 1995,

199/; Jiang et al., 1994; ThieJe et al, 2002).

It has been noted that the productivity has taken jump in India by the

development of many varieties since green revolution and these varieties

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Introduction 4

made a mosaic which reduced the pest risks (Fig.l). The recently released

varieties like PBW 343 etc. have IB/IR translocations thus have diverse gene

pool. The incorporation of rye segment into wheat could lead to a wonder

combination of traits and resulting high yields and wide adaptation. In fact,

alien gene transfers through wide hybridization, coupled with cytogenetic

technique, may be the alternative option for creating superior wheat

germplasm.

Fig 1: Genetic gain in yield after insertion of alien genes

1965 1970 1975 1980 1985 1990 1994 1995 1998 S227 K.Sona WL711 Arjun HD2329 CPAN3004 UP2338 PBW3A3 HD2687

Winter x Spring (1B/1R types types)

1.2 WIDE CROSSING: HISTORY AND PROSPECTS

Wide crossing altogether is not a very new concept. In 1876, when

Stephen Wilson humbly presented some completely sterile ears of wheat x rye

hybrid, a beginning was made. Despite this early successful effort, the

potential of wide hybridization in agronomic species was not appreciated until

after the rediscovery of Mendal's laws and chromosome engineering

techniques (Sears, 1972). First successful transfer of a single trait of

economic relevance from a related wild species into cultivated wheat was

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/nlrodiiclion 5

carried out by Sears (1956). He deliberately transferred a small insertion by

translocating a part of Ae. umbellulata to chromosome 6B of bread wheat

carrying resistance to leaf rust (Lr9). Later on by exploiting the homeologous

relationship between the genomes of related species and those of aesiivum and

durum wheat it became possible to incorporate genes or chromosome

segments from several of the related genera/species (Table 1).

Apart from the practical motivation to introgress genes from the alien

species into wheat, intergeneric hybrids offer promise to provide basic

cytological. evolutionary or phylogenetic information about the parental

species (Jauhar and Joppa, 1996; Bommineni et al., 1997). These wide crosses

are also a means of producing haploid plants by preferential chromosome

elimination of the pollen parent in early stage of development (Laurie and

Bennett, 1986; Inagaki and Mujeeb-Kazi. 1995).

During the evolution of the Triticeae the basic primeval genome of

seven pairs of homologous chromosomes has been modified following

speciation and allopolyploidization. However, a degree of similarity or

"homoeology' (Huskin, 1931) has been retained, so the genetic material may

be transferred to T. aestivum directly from closely related species with one or

more genomes in common with bread wheat. The triplication of much of the

genetic material in hexaploid i.e. AA, BB and DD (Sears 1954, 1966)

provides two advantages to alien gene transfer. Firstly, the wheat can tolerate

the loss or addition of whole chromosome usually without drastically

affecting the viability of the plant (Merker, 1992; Jauhar, 1993), and

secondly, duplicate genes present in the other two genomes can often mask

the effects of a deleterious genes present on an alien chromosome substituted

for a given wheat chromosome from the third genome. These factors coupled

with cytogenetic technique lead to the transfer of a number of desirable genes

into cultivated wheat, and set wheat ahead from other cereal crops in terms of

the possibilities for the introduction of genetic material from other species

(Ceoloni, 1987; Jiang el al., 1994).

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Introduction 6

Table 1: Alien wheat sources for specific traits

Traits Species

Leaf and stripe rust

Karnal bunt

Foliar blights

Moisture stress

Drought tolerance

Heat tolerance

Salt tolerance

Herbicide resistance

Apomixis

Male sterility

Biomass

Harvest index

Grain number

Grain weight

Grain quality

Lysine content

Storage protein

T. monococcum, T. boeticum, T. urartu, Aegilops

bicornis, Ae. speltoides, Ae. squarrosa, Ae. ovala,

Ae. triaristata. Ae. lorentii

T. boeticum, T. urartu, Ae. tauschii, T. araraticuin,

Ae. caudate, Ae. sharonensis

T. spelta, T. dicoccum

T. timopheevii

Ae. kotschyi, Ae. umbalullata, Ae. squarrosa

Ae. speltoides, Ae. triuncialis

Agropyron, Elynius, Secale cereale

Ae. bicornis, Ae. cylindrical

Elymus spp.

Ae. squarrosa

T. monococcum, T. dicoccum, Ae. speltoides.

Ae. Tauschii

T. monococcum, T. boeticum, T. urartu, Ae. bicornis,

Ae. tauschii

T. turgidum

T. turgidum, T. carthlicum, T. tlicoccoides

T. monococcum, T. boeticum, T. urartu, Secale cereale

T. boeticum, T urartu

T. dicoccoides

1.3 WILD TRITICUM AND GOAT GRASS RESERVOIR

A USEFUL

Extensive use has been made of a number of wild species in Aegilops

and Triticum (Gale and Miller 1987), high degree of homo/homoeology

between Triticum aestivum and its diploid and triploid progenitors and

Aegilops species make it an excellent material to cross with. They are also

sources of A, D and B genomes and constitute a rich repository of desirable

genes (Table 1). Therefore, the probability of introducing alien variation from

these genomes is much higher than from others (Kimber, 1993). Wild

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Introduclion 7

Trilicum and Aegilops, both are annual, divided into different sections ranged

from diploid to octaploid in some cases. In literature, it has been shown that

a number of useful genes have been extracted from them till date (Table-4).

Siill ihey have a potential to be taken as the genetic stock for the specific trait

improvement program or pyramiding the desired genes.

1.4 CONSTRAINTS TO WIDE CROSSING

I he challenge is to incorporate this "new" germplasm routinely into

existing wheal crop. The ways and means of exploiting them are depicted in

fig. 1. Hybrid production occupies the initial critical step as generally various

barriers encountered during production of wide crosses frustrated such

jtlempts. Some times the strong crossing incompatibility makes direct

h\ bridizatii)ii between wheat and other Trilicum and Aegilops species

difficult. Also in most of the cases of interspecific and intergeneric hybrids,

seed setting is very low as either embryo aborts at certain stage of

de\elopment or their subsequent development is arrested. The reason to

degeneration of embryos prior to full maturity may be (1) the inability of the

endosperm to carry out its normal role in supplying nutrients to the

developing embryo, or (2) the maternal tissue being hostile or antagonistic to

the development of the embryo. In other words embryo culture is useful when

the embryo has the ability to mature but is prevented from doing so (Ceoloni

ci a/.. 1993). In others, embryo does not abort but seed setting is very low. In

such cases, in vitro clonal propagation of hybrid embryos can generate more

plants to be used for further studies (Vasil, 1990).

The successful rescue of embryos paved the way for a series of

application of this technique to recover interspecific hybrids in a large

number of genera including Hordeum, Triticale, Agropyron and Trilicum.

The important examples of intergeneric hybrids obtained via embryo rescue

are barely x rye (Fedak 1986). wheat x barley (Koba et al., 1991), wheat x

maize (Lam-ie el al., 1990), Hordeum x Elymu.s (Lu and VanBothmer 1990;

Balyan and Fedak 1991), Elymus x Trilicum (Lu and Vanbothmer. 1990), T.

aeslivum x Aegilops (Ter-kuile el al. 1988), T. durum x Aegilop.s (Djenadi.

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Introduction 8

2000) etc. Disease resistance has been successfully introduced from Triticum

timopheevi to bread wheat via embryo rescue technique (Bhowal et al., 1993).

Embryo rescue was also used to obtain seeds from T. durum x T. timopheevi

(Claesson e /a / . , 1990).

Land Races - Wild Species Obsolete cv. Present cv.

Traditional Multiple Crosses

Wide Crossing Embryo Rescue

Protoplast fusion

New Genetic Variation

Wide Base Population Breeding Lines

Cycles of Recurrent Selection at Different Locations

In vitro selection , somaclonal variation, gametocedal variation,

transgenosis

Varieties

Fig.2: Possible strategy for exploitation of genetic resources and advanced genetic technique in a long term breeding programme (after Porceddu et al., 1988)

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Introduction 9

Although a number of other examples can be cited, it suffices to

emphasize that embryo culture, an easy and old tool, has not been fully

exploited. More efforts should be made to employ this method for obtaining

intergeneric and interspecific hybrids, and for increasing the genetic diversity

in wheat.

1.6 STUDY UNDERTAKEN

Agronomically useful hybrids, even between closely related species,

are often unattainable because of sexual incompatibility. In many instances

fertilization takes place, but the developing embryo aborts at various stages of

development. The technique of embryo rescue which involves the excision

and culture of the young hybrid embryos, has proven to be very useful in

growing such embryos to maturity and in obtaining hybrid plants.

Furthermore, genetic and field evaluation of the hybrids can be accelerated by

generating multiple plantlets of the embryos in culture.

Keeping the above in view the following objectives were taken to:

1. standardize the regeneration protocol of immature embryos and

hardening procedure of the regenerants.

2. selecting the better responsive genotypes of both aestivum and durum

wheat to be used as female/recipient plant in the crossing program.

3. attempt the crosses with alien donor plant

4. find out the time to scoop embryos from hybrids.

5. culture rescued embryo in vitro.

6. develop full plant from hybrid embryos which otherwise aborts

normally.

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RBt/I£^ OV^ UTBkATUtiM

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2. Review of Literature

Among the cultivated forms of wheat ^Triticum aestivum L.em. Thell.

(2n=6x=42,BBAADD) and T. durum desf (2n=4x=28, BBAA)] are natural

allopolyloids. Their cytoplasm is known to come from B genome (Ogihara and

Tsunewaki. 1988; Dvork and Zhang, 1990) and donor of B genome is similar to

Aegilops speltoides Tausch. (Sarkar and Stabbins, 1956). At times species of the

Aegilops, section sitopsis such as Ae. searsii, Ae. longissima or Ae. sharonesis was

also considered as a possible source of B genome (Kimber and Feldman, 1987). The

source of A genome is now thought to be T. urartu Tum. rather than T. monococcum

L. ssp. boeticum Boiss. (Waines et al., 1993). The source of D genome is Ae. lauschii

Coss, more commonly called Ae. squarrosa L. in the literature (Kihara, 1924, 1944;

McFadden and Sears, 1944). The wild progenitors of cultivated wheat comprises a

rich repository of the desirable traits which could potentially be transferred into

cultivated wheat through sexual hybridization (Harlan, 1976; Plucknett et ai, 1987;

Lange and Balkema-Boomstra, 1988; Ceoloni et al., 1993). However, due to cross

incompatability in many of the wide crosses either embryo gets aborted or seed

setting is very low. By aid of embryo rescue a sufficient number of progenies can be

obtained to use for further studies (Vasil, 1990).

The literature reveals that the in vitro culture response of Triticum is very

much genotype specific (Maddock et al. 1983, Machii et al, 1998). Although several

other factors can affect the callus induction and plant regeneration from the rescued

embryo, if we have a better responding genotype as one of the parents, the chances of

getting regenerants becomes high (Ou et al, 1989). Therefore, screening of the wheat

genotypes for culture responses and having a reliable protocol is a prerequisite before

we go for embryo rescue of wide crosses. Keeping these facts in view, the current

research problem is being reviewed imder three major aspects of embryo culture, wide

hybridization and gene transfer.

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Review of Literature 11

2.1. EMBRYO CULTURE

Since the early reports on plant regeneration from wheat callus by Adachi and

Katayana (1969) and Shimada et al. (1969), the techniques have been continuously

improved. Although wheat was previously thought to be recalcitrant, recent work has

established that immature embryos of wheat form embryogenic callus in a

reproducible manner and multiple plants can also be obtained from these calli.

Regeneration of plants from callus cultures occur by one of the two pathways,

embryogenesis and organogenosis. In wheat, regeneration by both pathways is

possible in same callus (Mathis and Boyd, 1986; Mathias, 1990; Kothari et al., 1998).

Organogenic type of regeneration was reported in early experiments (Chin and Scott,

1977; O'Hara and Street, 1978; Sears and Deckard, 1982). Histological investigations

have also shown the existence of organogensis in both Triticum aestivum (De Buyser

et al. 1988; He, 1989) and in T. durum (Bennici et al, 1988). Shoots were also

reported to originate from "Green spots" (Ivanov et al, 1998, Machii et al, 1998).

Embryogenic callus of wheat can be characterized by glistening pale yellow surface

and compact appearance, sometimes may be white and opaque which later on may

become nodular with pale yellow and green buds. On this type of white callus,

formation of typical embrj'oids of wheat were first observed by Ozias-Akins and

Vasil(1983a,b).

Somatic embryos or embryoids are morphologically and physiologically

similar to zygotic embryos (Scott et al, 1990). Some workers (Haccius, 1978; Vasil

and Vasil, 1982) consider that embryoids like zygotic embryos originate from single

cells and thus if mutation has occurred or exogenous gene have been introduced,

whole regenerants are genetically modified. However, others (Werinicke et al, 1982)

have proposed that embryoids are broadly based and hence of multiple cell origin.

Nonetheless, the multiple celled proembryogenic tissues may still have originated

from a single cell (Maddock, 1985; Williams and Maheshwari, 1986). In contrast,

organogenesis may produce chimeras and thus formation of plants through

organogenesis are not very much preferred for clonal propagation, mutation research,

genetic analysis, transformation and breeding (Vasil, 1987).

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Review of Literature 12

Embryogenesis in wheat is known to be affected by genotype (Chu et ai,

1984; He et ai, 1986), phytohormone concentration and type (Hunsinger and Schauz,

1987) and interaction between hormone and sugar concentration (Ozias-Akins and

Vasil, 1983b). It can be greatly enhanced by culturing on two fold concentration of

MS medium (Ozias-Akins and Vasil, 1983b), on medium containing a high

concentration of salts (Galiba and Yamada, 1988; He et al, 1989) or on medium

without zinc (He, 1989). However, the relative frequency of embryogenesis,

organogenesis and the effect of explant, media, and culture conditions, on the balance

between the two pathways are unclear. The salient information available in literature

on various factors influencing in vitro response of immature embryos of wheat has

been reviewed in following table.

Table 2 : Factors influencing in vitro responses of immature embryos of wlieat

Factor Age (daj 10-14 10-15

16-22

19-20 >22 Up to 25

Used to {S post pollination)

Optimum age

fast growing and regenerable calli

85% callus and regeneration optimum age precocious germination callusing on medium having 2,4-D -i- ABA

Reported by

Wang et ai, 1993; Arun et ai. 1994

Shimada, 1978; Sears & Deckard, 1982; Magnussan & Bomman, 1985; Ozygen et al, 1998

Zhang, 1986

Karadimova e/a/., 1985

He era/., 1988 Quarashi e/a/, 1989

Size of embryo (mm) ~ 1.0 mm

1.0-1.5mm

>0.5-2mm

0.9-1.8 <0.9-1.8

optimum callus

better callus

no effect on callus morphogenesis, affect rate of morphogenesis epiblast callus scutellum callus

Ozias-Akins & Vasil, 1982; Bohorova er a/., 1995 Liang et al, 1988; Redway et a/., 1990 Maddock etal, 1983

He era/. ,1986 He era/., 1988

Orientation of embryo Scutellum upward check precocious germination Scutellum downward

Eapen & Rao, 1985; Ivanov et al, 1998

Hcetal, 1986

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Review of Literature 13

Basal culture medium MS

B5 N6 LS P-medium

Hussain and Islam, 1993; Ganeva et al, 1995; Zair et al, 1995; Kothari et al., 1998; Ozygen et al, 1998; Karaca & Burun, 1999 Chawla & Wenzel, 1987; Tuberosa et al, 1988; Chawla, 1989

Jia & Zhao, 1993; Bohorova et al, 1995 Gaponekoe/a/., 1985a, b; Heyser e/a/., 1985 hazQxetal, 1983; Sharmae/a/., 1998

Phytohormones 2,4-D(0.1mg/l) 2,4-D (0.5 mg/1) 2.4-D(l mg/1) 2,4-D (1 mg 1.5/1) 2,4-D (2 mg/1)

2,4-D (2.5-5mg/l) 2.4-5-t + Kinatin

lAA (2.5 mg/1) NAA (2.5-5 mg/1) Dicamba

Picloram

NAA+BAP lAA

Kinetin

BAP

lAA+Kinetin lAA+BA

no callusing no callusing Callus Callus Callus

reduced callus with rooting Optrimum callus but reduced regeneration no callusing no callusing better than 2, 4-D for callus induction plantlets less vigorous and difficult to establish in pot

double amount of callus then on 2,4-D good callus and regeneration in regeneration medium induce rooting

no effect on callus induction inhibited callus induction

callus formation improve plant regeneration

induce plant regeneration

used in root formation regeneration and rooting as well

Heetal, 1986 Rashid & Quaraishi, 1989 Maddock etal, 1983 Abdrabou & Moustafa, 1993 Sears & Deckard, 1982,; Mathias et al, 1986; Ozyg en etal, 1998 Chauhan «& Singh, 1995 Lazeretal, 1983

Ozias-Akins & Vasil, 1982 Ozias-Akins & Vasil, 1982 Hunsinger & Schauz, 1987

Paperfuss & Carman, 1987

Lazeretal, 1983

Anshulika e/a/., 1999 Quaraishi, 1989

Ahloowalia 1982

Butonko & Dzhardemaliev, 1986 Lazeretal, 1983 Bajaj, 1985; Shrivastava et al, 2000

Mathias etal, 1988; Hussain & Islam, 1993 Ivanov et al, 1998 Bohorova e a/., 1995

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Review of Literature 14

Zeatin

Zeatin + lAA Auxin/2,4-D free basal medium Gbberelic acid (GA3) ABA (0.25 mg/1)

ABA (0.1 mg/1)

Ethylene

AgN03

inhibit callus induction promote growth reduce embryogenesis

improve shoot regeneration frequency inhibit root formation beneficial for shoot formation Regeneration

enhance rooting in callus no effect on regeneration

vigorous precocious germi­nation help in inducing callus increase embryogenesis little effect on callus induction strongly inhibit precocious germination inhibit shoot formation inhibit ethylene action

Ahloowalia, 1982

Carman, 1989

Gosch Wackerle et al, 1979

Tanzarella & Greco, 1985 Ozias Akins «& Vasil, 1982 Symillidese/o/., 1995; Taghvaie/a/., 1998 Ozias-Akins & Vasil, 1982

Machiie/a/., 1998

Brown era/., 1989 Morris et al, 1986; Quarashi etal, 1989

Papenfuss & Carman, 1987 Pumhauser e/a/., 1987

Organic additives Yeast extract Casein hydrolysate

Coconut milk

Potato extract

Aspergine Proline

Gultamine

Tyrosine

promote callusing

induce embryogenesis

suppress precocious germination promote initiation, growth and regeneration of callus promote callusing no significant improvement better callus induction

prolong totipotency better callus induction

root formation improve regeneration potential

regeneration medium

Zamora & Scott, 1983 Shimadaero/, 1969

Ozias-Akins & Vasil, 1983b Ahloowalia, 1982; Mathias & Simpson, 1986 Zhaire/a/., 1995

Karaca & Burun, 1999 Machii et al, 1998; Malhotra et al, 1999

Tuberosa e/a/., 1988

Sharma et al, 1998; Anshulika etal, 1999 Ivanov era/, 1998

Shrivastava et al, 2000; Shrivastava & Chawla, 2001 Hussain & Islam, 1993

Carbohydrate Sucrose

Glucose Maltose

(2%) Symillides et al, 1995; Ivanov et al, 1998; (3%) He et al, 1989; (9%) Sharma et al, 1998

(2%) Ozias-Akins & Vasil, 1983a (6%)Machiiera/, 1998

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Review of Literature 15

Gelling agent Agar Agarose Gelrite Liquid medium

(0.8%) Jauhar, 1991; (0.7%) Ivanov etal., 1998

(0.6%) Inagaki & Tahir. 1990a; Symillides et ai, 1995 (2%)Machiie/a/., 1998 Dolezel er a/.. 1980

Effect of environmental conditions on culture establishment Growth condition of explant plant have a significant effect on culture response

Larkin et al, 1984; Carman et ai, 1988

No marked effect Mathias, 1990 Green house grown embryosshows poor response Purnhauser e/a/., 1987

Growth condition of cultures Dark during callus induction Light during callus induction

Dark during regeneration phase Light during regeneration phase

calli grow faster and morphogenic decrease regeneration potential for longer time

promote precocious germination

promote regeneration

Papenfuss & Carman, 1987; Higgins & Mathias, 1987 He et al, 1986; Lazer et al., 1983

Mathias etal., 1986

Ozias-Akins & Vasil, 1983c

Genotype Influence callus induction Influence callus growth rate Frequency of embryogenesis Plantlet regeneration frequency

Mathias & Simpson, 1986; Machii et al., 1998 Lazer etal., 1983 Arun et al., 1994; J'Aiti et al., 1999

Karadimova et al., 1985

Cytoplasm Influence in vitro response Mathias & Fukui, 1986

Genie control stimulate callus growth and embryogenesis

not a sole factor to better culture response 6 & 7 chromosomes of Secale cereale induce higher regeneration frequency 7B, 7D and ID are important to regulation Rhtl and Rht2 loci, modify callus growth and embryogenesis

Langridge et ai, 1991; Fennell et al., 1996 Henry Buyser, 1990 Lazer era/., 1987

Langridge er a/., 1991 Mathias & Atkinson, 1987; Yamamoto & Miuri, 1995; Franzone et al., 1998

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Review of Literature 16

2.2. WIDE HYBRIDIZATION

The history of hybridization of alien species with Triticum goes back

to 1876, when Wilson made the first hybrid involving wheat x rye. However,

Rimpau (1891) obtained seeds in a wheat x rye hybrid to represent the first

triticale. The systematic work on wide hybridization was started only after the

first decade of last century when many hybrids involving Triticum and

Aegilops species were produced. The pioneering work of Anton Kruse in

attempting hybridization with Triticum aestivum x Avena sativa (Kruse,

1969), Hordeum vulgare x Secale cereale (Kruse, 1967) and H. vulgare x T.

aestivum (Kruse, 1973) played an important role in accelerating the research

in the area of intergeneric hybridization involving T. aestivum or T. durum

with species of Agropyron, Aegilops, Elymus, Haynaldia, Hordium etc.

2.3. CONSTRAINTS TO WIDE HYBRIDIZATION

Assortments of barriers are inherent in an attempt to utilize exotic

germplasm for crop improvement. Successful sexual hybridization involves a

sequence of events including pollen germination, pollen tube growth,

fertilization, embryo and enodsperm development and seed maturation.

Stebbins (1950) has divided these hybridization barriers into two main groups

namely, pre-fertilization and post-fertilization barriers. The pre-fertilization

category includes external barriers like geographic isolation and internal

barriers like apomixis, pollen pistil incompatibilities and prevention of

fertilization. Post-fertilization barriers are a greater hindrance to

hybridization and can be a result of ploidy differences, chromosome

alterations, incompatible cytoplasm, seed dormancy and hybrid breakdown

etc. These barriers can be overcome by making alterations in choice of

parents, emasculation procedure, pre and post pollination treatments and

embryo excision and culture (Mujeeb-Kazi and Kimber, 1985).

Laibach (1925) was the first to use embryo culture successfully in

cross of Linum austriacum x L. perenne. Since then several refinements have

been made in embryo culture/embryo rescue technique. It paved a way to the

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Review of Literature 17

successful hybridization of a number of otherwise incompatible intergeneric

and interspecific crosses. Currently embryo rescue holds great promise not

only for effecting wide crosses but also for obtaining plants for inherently

weak embryos, obtaining haploid plants as well as shortening the breeding

cycle and overcoming hybrid necrosis.

2.4. STRATAGIES TO EXPLOIT ALIEN SOURCES

2.4.1. CHOICE OF PROMISING AND COMPATIBLE PARENTS

Introgression of alien variability is demarcated into two distinct areas

of long-term intergeneric and short-term interspecific hybridizations. These

are separated on the basis of wheat/alien genomic similarity and level of

genetic recombination. Interspecific approaches are favored for genetic

introgression (Mujeeb-Kazi and Asiedu. 1995). When more diverse or distant

hybridization were initiated, hybrid production constraints were increased

manifold and arrays of manipulative techniques were required. Moreover,

wider the crosses are, lesser the stability of the transferred gene (Dhaliwal et

al, 1993). Additionally, genotypes, ploidy levels, cross direction and

environment all become contributors to hybrid production success (Sharma

and Gill, 1983; Mujeeb-Kazi and Kimber, 1985). A hybrid production

procedure in which wheat is the maternal parent is being routinely adopted by

various authors with significant success (Mujeeb-Kazi et al, 1987).

Alien diploids have been difficult to hybridize with wheat. Such

crossability barriers can be overcome either by reciprocal crosses or by first

doubling the alien diploid and using the induced autotetraploid as the pollen

parent (Mujeeb-Kaziitfl/ji^dy 1995). Specific examples of intergeneric hybrids

produced by this procedure are: T. aestivum x A. cristanum (4x), T. aestivum

X Psathyrostachys juncea (4x), T. turgidum x P. juncea (4x), in which each

hybrid possesses 35, 35, and 28 chromosomes respectively.

Certain wheat cultivars are more crossable than others (Jauhar, 1993).

Crossibility of wheat and rye, for example, is controlled by two recessive

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Review of Literature 18

alleles Krl and Kr2 on chromosome 5A and 5B respectively (Riley and

Chapman, 1967), prevents their crossability with Secale cereale (Lein, 1943)

and Hordeum bulbosum L. (Snape et ai, 1979; Tanner and Falk, 1981).

Moreover, wheat cultivars that give high seed set in crosses with rye are

reported to produce high seed set after pollination with several species of

Triticum, Secale, Agropyron, Elytrigia and Elymus.

2.4.2. CROSSING PROCEDURE AND PRE/POST POLLINATION TREATMENTS

In most of the wide crosses, the wheat is generally used as female

parent, unless cytoplasmic characters have to be transferred. In routine, spikes

of wheat are manually emasculated and receptive stigma is pollinated at least

twice with the pollen of the donor parent. Some times hot water is being used

to emasculate the spikes (Inagaki et al. 1997). The spikes are immersed in

hot water set at 43°C for 3 minutes one day before pollination and pollinated

after cutting the upper part of the florets but leaving the anthers.

The emasculated florets are recommended to treat with growth

hormones or immunosuppresant (Kruse, 1967). The 1:1 mixture (v/v) of 2,4-D

(25mg/l) + GA3 (75mg/I), one day before and one day after pollination was

recommended to be used (Jauhar, 1991, 1993). A hormone solution containing

GA3 (75mg/I) + 2,4-D (5mg/l) was used by Jauhar and Peterson (1996) and

Bommineni et al. (1997) only one day after pollination. Riera-Lizarazu and

Mujeeb-Kazi (1993) used aqueous solution of 50mg/l 2,4-D + 150mg/l GA3,

only one day after pollination. However, Mujeeb-Kazi and Asiedu (1990)

used only GA3 (75 ppm aqueous) up to 4 days post pollination. These authors

recommended treatment of growth hormone by spraying the solution on the

pollinated spike or flooding the cup by hormone solution. While some other

researchers (Inagaki and Tahir, 1990b; Inagaki and Mujeeb-Kazi, 1995,

Inagaki et al, 1997) needle injected lOOmg/1 2,4-D solution in the upper

nodes of the wheat clum the pollinated spike. Even some of the other

researchers used 2,4-D in combination of AgN03 (O'Donoughae and Bennet,

1994; Almouslem et al, 1998), and reported it to give better seed set then

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Review of Literature 19

2,4-D + GA3 solutions. Almouslen e( al. (1998) also mist sprayed the spike

with tween-20 (1% v/v) as surfactant before spraying hormone solution.

2.5. EMBRYO RESCUE

To make interspecific and intergeneric hybrids with bread wheat, the

latter is generally used as the female parent and embryo rescue is practiced to

save the aborting embryos and to rear them to the seedling stage. It is a wider

term and includes immature embryo culture, ovule/ ovary culture and floret

culture. In some cases even spikelet and spike culture.

2.5.1. IMMATURE EMBRYO CULTURE

Hybrid embryos are rescued by aseptically removing embryos prior to

their abortion and culturing them directly onto an artificial media. An

important aspect of embryo culture is a priori knowledge of the in vitro

system for the species of interest and has a suitable culture system for the

species being hybridized (Collins et al, 1993).

2.5.1.1. Stage of scooping out

The successful rescue is influenced by the age of the embryo at the

time of its culture. Two basic stages of the embryo growth exist with regard

to nutritional independence i.e. heterotrophic and autotrophic stage

(Raghavan, 1976). The heterotrophic stage of growth is the period during

which embryo depend on the endosperm for its nutrition and extends from

fertilization up to approximately the heart stage and termed as pro-embryo.

During the heterotrophic stage, the embryo usually requires presence of

growth regulators to allow for its proper development (Raghavan and

Srivastava, 1982). The autotrophic phase of growth begins at the late heart

stage. This stage is significant for in vitro culture, as embryo becomes

sufficiently independent from endosperm for its further growth. The growth

requirements for artificial culture for these embryos are minimal and also they

can withstand the isolation shock (Collins et al, 1993). The culture of young

embryos is also more difficult due to their size, damage during dissection and

sensitivity to osmotic change (Maheshwari and Rangaswamy, 1965).

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Review of Literature 20

The Stage at which embryos can be rescued depends on the time at

which endosperm aborts and the time at which subsequent embryo

degeneration results. For example. Gill et al. (1981) recorded that in cross of

Aegilops squarrosa x T. boeticum, endosperms aborted at about 10 days and

embryos aborted by 14 days following pollination. Hybrids were obtained by

rescuing embryos 10 days after pollination.

2.5.1.2. Culture pathways

There are two different pathways to be followed for culture of rescued

embryos. One is by going through direct germination of embryos and second

through dedifferentiation and subsequently regenerating the whole plant.

Alien gene transfer mediated by callus culture promotes multiple cytological

variations. The alien exchanges through callus culture have been

demonstrated in wheat x rye hybrids (Lapitan et al, 1984, 1986) and are

being applied to other intergeneric hybrids {T. aestivum x Ae. variabilis) with

poor F2 wheat/alien chromosomal recombination (Ter-Kuile et al., 1988).

Amphipliods of intergeneric hybrids represent an important means of

germplasm distribution and organized alien genetic component exploitation,

particularly for stable amphiploids. It has been rather difficult to induce

amphiploidy in several intergeneric hybrids by colchicine treatment. In an

observation by Ter-Kuile et al. (1988), it has been anticipated that the

regenerated Fi hybrids of T. aestivum x Ae. variabilis and T. turgidum x Ae.

variabilis demonstrated seed set in an otherwise self sterile population. Callus

induced doubling may prove advantageous for combinations otherwise hard to

double (Mujeeb-Kazi and Asiedu, 1990).

2.5.1.3. Media manipulation

The successful production of plants from the cultured embryos largely

depends upon the maturation stage and composition of the medium. Culture

medium requirements are almost same for rescued embryos as for normal

immature embryos. Some authors recommend amino acids (organic nitrogen)

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Review of Literature 21

rich LS/MS medium to be used for culturing very small rescued embryos

(Valkoun et al, 1990). Most culture media include an inorganic source of

nitrogen, however, the addition of an organic form has been shown to have a

stimulating effect on embryo growth and development (Monnier, 1978).

The form in which organic nitrogen is supplemented is also important.

Although aspergine is found to be beneficial in some experiments (Lea et al.,

1978), glutamine is generally superior for a wide range of genera (Mok et al..

1978). However, glutamine alone does not always give the best response. A

mixture of amino acids may be more effective in some instances especially

for very small embryos. An undifferentiated nitrogen source such as casein

hydrolysate, coconut milk, potato or yeast extract (Bajaj, 1990) has been

shown to be beneficial.

MS is the most extensively used medium for rescuing embryo

(Gaponeko et al., 1985a,b). Orchid agar (Jauhar, 1991, 1995), Taira and

Lartor's (1978) medium (Mujeeb-Kazi and Asiedu, 1990), Lanssmier and

Scoog (1965) medium (Gaponenko et al., 1985a; Valkaun et al., 1990) have

also been used.

2.5.6. ALTERNATIVE METHODS

Embryos were also reported to culture on the endosperm of other

species or one of the parent's endosperm to rear them to plantlet. This method

helped to obtain a number of intergeneric & interspecific crosses like

Hordeum x Triticum (Kruse, 1973, 1974a, b; Williams and De Lautour, 1980,

1981; Williams & Williams, 1983). In some other, where technically it is not

possible to scoop out an embryo, ovule culture (Stewart, 1981; Comeau et al.,

1988, Agnihotri, 1990; Nikova and Zogorska, 1990), spikelet culture (Zhang

et al., 1996) or even spike/detached tiller culture (Inagaki et al., 1997;

Monika ,1999; Cherkaoui et al., 2000) were deployed.

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Review of Literature 22

2.6. ALIEN GENES TRANSFERRED IN WHEAT

Since the report of Kruse (1967, 1969, 1973, 1974) there has been

heightened interest world wide in the production and exploitation of

interspecific/intergeneric hybrids. The enormous array of genetic stock

available in secondary gene pool of Triticum has been tapped for a number of

desirable characters. Some important ones are enlisted in following table.

Table 3: Disease resistance genes incorporated in wheat gene pool from alien sources.

Gene Donar plant Reported by Resistance to Puccinia graminis Disease : Black rust, stem rust Sr2 Sr9e Sr l3 Sr l4 Srl7 Sr24 Sr25 Sr26 Sr27 Sr31 Sr37 Sr38 Sr43

T. dicoccum T. dicoccum T. dicoccum T. dicoccum T. dicoccum A^ropyron elongatum A. elon^atum A. elon^atum S. cereale S. cereale T. timopheevii Ae. ventricosa A. elongatum

Hare & Mcintosh, 1979 Luig& Watson, 1967 Knott, 1962; Mcintosh e/a/., 197?-Knott, 1962 E. S. Macfedden (unpublished) Sears, 1973; Mcintosh et al., 1977 Sears, 1973; Mcintosh e a/., 1977 Mcintosh era/., 1977 Acosta, 1963; Marais & Marais, 1994 Friebee/o/., 1996 Mcintosh & Gyarfas, 1971; Gyarfas, 1978 Banana etai, 1969 Kibirige-Sebunya & Knott, 1983; Kim et al., 1993; Friebeera/., 1996

Resistance to Puccinia recondite Disease : Brown rust/ leaf rust Lr9 Lrl9 Lr21 Lr22a Lr24 Lr25 Lr26 Lr28

Ae. umbellulata A. elongatum Ae. squarrosa Ae. squarrosa A. elongatum S. cereale S. cereale Ae. speltoides

Sears, 1961, 1972 Browder, 1973a Rowland & Kerber, 1974 Dyck & Kerber, 1970; Kolmer, 1997 Browder, 1973b Heun and Friebe, 1989 Mettin, 1978 Mcintosh er a/., 1982

Resistance Puccinia striiformis Disease : Stripe rust/Yellow rust Yr5 Yr8 Yr9 YrlO Yrl5

T. spelta Ae. comosa S. cereale T. spelta T. dicoccoides

Macer, 1966 Riley era/., 1968 a, b Zeller, 1973, Mettin et al., 1978 Kema&Lange, 1992 Gerechter-Amitai et al., 1989

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MATBtOAUANP MBTHOPS

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3. Materials and Methods

3.1. PROCUREMENT OF WHEAT GERMPLASM

Literature reveals that in vitro response of wheat for

regeneration is genotype specific. Therefore, it is a prerequisite to

establish a reliable protocol for regeneration of genotypes to be used in

wide hybridization program. A range of varieties of both bread as well

as durum wheat (Table 4) were selected by keeping in view the best

variety available and acreage covered. Seeds were provided by

Germplasm Resource Unit, Directorate of Wheat Research, Karnal

India, and were sown in polyhouse under controlled environmental

conditions in three rows plot.

Table 4:Varieties used in standardization of regeneration protocol from immature embryos and selection of genotypes to be used in wide crossing.

Bread wheat Durum wheat

1.CPAN3004 1. Bijaga Yellow

2. HD 2329 2. Gulab

3. KRL 1-4 3. HD4530

4. PBW 343 4. HI 8381

5. PBW 373 5. MACS 2846

6. Raj 3765 6. PDW 215

7. UP 2338 7. PDW 233

8. WH 542 8. Raj 1555

Wide Hybridization Program

After screening the genotypes, three varieties each of bread and

durum wheat (Table 5) were selected to use as a female parent in the

wide crossing program. Varieties were sown in staggered manner at 10

days interval in five rows each to maintain the continuous availability

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Materials and Methods 24

of female plant. Five sowings were done by starting in first weak of

November.

Table 5: Varieties selected for wide crossing program

Sr. No. Bread wheat Sr. No. Durum wheat

1.

2.

J .

4.

5.

WH 542

UP 2338

PBW 343

Raj 3765

HD 2329

1.

2.

J .

4.

5.

Raj 155 5

Bijaga yellow

Gulab

PDW 215

PDW 233

3.2. ALIEN GERMPLASM

Many accessions of diploid and tetraploid Triticum and Aegilops

species were used for alien gene resource taken as a male donor plant

in the wide crossing program (Table 6).

Table 6: Wild species used in crossing program

Sr. Species No.

Accessions Sr. available No.

Species Accessions available

2.

J .

4.

5.

6.

7.

8.

9.

10.

T. urartu (A")

T. boeticum (A )

Aegihps speltoides (S)

Ae. longissima (S')

Ae. sharonensis (S " )

Ae. bicornis (S )

Ae. squarrosa (D)

Ae. caudate (C)

Ae. markgrafi (C)

Ae umbellulata (U)

17

2

44

2

2

7

87

2

2

2

11

12

13

14

15

16

17

18

19

20

Ae. cylindrica (CD) 30

Ae. ventricosa (D") 1

Ae. kotschyi (US) 4

Ae. peregrina (US) 5

T. triuncialis (UC) 34

Ae. ovata (UM°) 9

Ae. lorentii ( U M ' ' ) 3

Ae. triaristata (UM') 11

Ae. geniculata (UM°) 2

T. araraticum (AG) 1_

Seeds were procured from Germplasm Resource Unit, Directorate

of Wheat Research, Karnal India. The wild species were grown in net

house, maintaining 2.0 ft distance between rows to avoid any

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Materials and Methods 25

contamination and seed dispersal. Additional photoperiod of six to

eight hours was provided with bulbs of lOOOWt to facilitate flowering

of alien species. To break the dormancy, the seeds were stored at -4°C

for vernalization.

3.3. HARVESTING OF IMMATURE EMBRYOS AND WIDE CROSSING 10 to 18 days old embryos were cultured in the present study to

evaluate the optimum age of embryos. Only primary florets from the

central portion were left on the spike to be used for excision of

explant. Rests of the florets along with awns were removed before

taking the spike to the laminar flow bench.

The spikes quarter to half came out from flag leaf were used for

hand emasculation. The upper and the lower spikelets were plucked out

and from the left outs the secondary and the tertiary florets were also

removed. The spikelets were than cut slightly below the center with the

help of sharp scissors. Anthers were removed by forceps avoiding any

injury to the gynoecium. Utmost care was taken not to leave any anther

and secondary or tertiary floret to avoid selfing. The spikes were then

covered by butter paper envelope to secure humidity and resist

undesirable pollination. When the stigma became feathery, pollination

was done for two consecutive days. Pollinated spikes were sprayed by

0.05% 2,4-D + 0 .1% GA3 solution in the evening, for four days starting

by the day the spike was emasculated.

The crosses were made in the replication of 20-25 spikes per

cross in five days i.e. 3 to 4 spikes every day for each cross

combination. The spikes were continuously examined and three days

prior to the day the florets starts getting dried, were recorded as the

day of abortion of the embryo. The embryos were rescued from the rest

of the spikes accordingly. For making replications each spike was

divided into three and these three portions were taken at alternate days.

Three spikes per cross was left in the field as control.

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Materials and Methods 26

3 . 4 . In vitro C U L T U R E

3.4.1. BASAL CULTURE MEDIUM

3.4.LI Immature embryo culture

Growth and morphogenesis of plant tissues in vitro are largely

governed by the composition of culture media. In the present study, the

revised MS medium (Murashige and Scoog, 1962) and the B5 medium

(Gamborg et al., 1968) were used as the basal medium in

standardization of the medium for embryo culture along with different

hormonal combinations. The details of the ingredients along with

recipe for the preparation of stock solutions of these mediums were

provided in annexure.

3.4.1.2. Rescued embryo culture

Two different paths were adopted to culture rescued embryos,

one through direct germination of embryos and another through

dedifferentiation into callus. To induce callus MS + 2,4-D (2 mg/1). For

regeneration, Rl method, used with normal immature embryo i.e., left

on the same medium and exposed to light provided by 20 Wt. electric

bulbs (Philips India). Calli were also transferred on the CM, EC and

then on R2 or R5 (Table 7) to get regeneration. Regenerated plantlets

were transferred on S3 and finally plants with two to three leaves were

transferred on Rt4 for root development (table 4).

3.4.2. GROWTH REGULATORS

2,4-D alone or in combination with lAA was used to induce

callus. Kinatin and BAP alone or with lAA were used to achieve

regeneration. Kinatin/Kinatin+GAs helped in elongation of regenerants.

NAA/IAA was used to get healthy roots. Preparation of stock solutions,

storage, sterilization procedure etc., was provided in annexure.

3.4.3. PREPARATION OF CULTURE MEDIUM

To prepare one-liter culture medium, 250ml double distilled

water (ddw) was taken in conical flask. All the ingredients were added

26

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Materials and Methods 28

to it according to table 4, except agar and final volume finished to

600ml. pH was adjusted to 5.8 by adding IN NaOH/1 N HCl. In another

one liter flask 8gm agar was dissolved in 400ml water. This melted

agar was than mixed to the 600 ml solution and homogenized by

stirring. In test tubes measuring 150 x 25mm, 15ml of the medium were

dispensed while in Erlenmeyer flasks (100ml capacity) 40-45 ml of the

medium was dispensed. The culture vessels were plugged by non-

absorbent cotton wool wrapped in single layered muslin cloth. Medium

and accessories were sterilized by autoclaving at 121°C temperature

and 15 psi pressure for 15 minute. The medium was autoclaved in

aliquots of 250 ml in one liter flask for 20 minutes when to be used in

petridishes. After autoclaving, the medium, in the flasks was allowed

to solidify as such and slants were made in test tubes. For pouring in

petridishes the medium were cooled to 50°C and poured in sterilized

petridishes on laminar flow bench. The autoclaved and solidified

medium was left on room temperature for four to five days to check

microbial contamination and afterward stored in refrigerator. This

media could easily be used till one month.

3.5. ASEPTIC TECHNIQUE

The aseptic techniques are necessary to create aseptic

environment to avoid contamination. The technique involves

sterilization of working bench, glassware, instruments, plant material

inoculation and incubation. Laminar airflow hood were sterilized by

UV-lights for one hour and wiping the surface with 70% ethanol before

any operation under the hood. The glassware, blotting sheets, cotton

wool etc. to be used in different processes were wrapped in butter

paper or aluminium foil and sterilized by autoclaving at 15 lbs/inch^

pressure (121°C) for 20 minutes.

3.6. STERILIZATION AND PLATING OF PLANT MATERIAL

The spikes were surface sterilized by 70% ethanol (v/v) and

taken to the laminar airflow cabinet. The seeds were threshed out on

28

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the laminar and treated with freshly prepared 0 .1% solution of mercuric

chloride plus a drop of Tween 20 for 2 to 5 minutes, followed by a

quick wash in 70% ethanol and 6 to 7 rinses by autoclaved distilled

water. The embryos were scooped out aseptically under the Stereo-

zoom microscope taking utmost care that no piercing or injury occur to

the embryos and plated on the culture medium with scutellum side

upward.

3.7. INCUBATION OF CULTURES

Temperature was maintained on 25 ± 2°C during entire course of

study with a relative humidity of 50 to 60%. Calli vv ere incubated in

dark. The diffuse white lights were provided by 100 |j,mol m"'S"'Wt.

Philips, India cool tube rods and yellow lights by 20 Wt. Philips, India

bulbs.

3.8. HARDENING OF THE PLANTLETS

Plants with well-developed white roots were kept on hardening

to transfer into field. Different procedures were assumed.

> The plantlets were first transferred to the liquid medium. The test

tubes were covered by poly bags enriched with humidity by

spraying water. Established ones were transferred to soil mixture

containing equal proportions of soil, farm yard manure (FYM) and

sand, in growth chamber at 70 to 90 relative humidity and 25 ± 2°C

temperature.

> The plantlets grown on the root induction medium were transferred

to MS half strength medium + 1 0 mg sucrose + 6 g agar and then to

the '/4 MS medium + 2 gm sucrose + 4 g agar. Further plants were

transferred to liquid medium supported by blotting papers. The

established plants were transferred to soil mixture containing equal

amount of soilrite, perlite and soil.

> The plantlets with developed white roots were taken out of the agar

medium and washed carefully so as to remove every trace of media.

29

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Materials and Methods 30

Plants were than transferred to 1:1 mixture of agropeat and soilrite

plantlets were irrigated by MS medium containing inorganic

constituents only. The plants were kept in growth chamber at 1 5 ±

1°C temperature and 80 to 90% relative humidity till two to three

fresh leaves emerged out. Thereafter these were transferred into the

field/bigger pots containing soil, sand, FYM and agro peat in equal

proportions.

30

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RBSUITS

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4. Results

EXPERIMENT 1

4.1. STANDARDIZATION OF REGENERATION PROTOCOL

Extensive studies were carried out on the effect of different basal culture

media, growth regulators and age of embryos (days post pollination, DPP) on the

initiation and growth of callus and regeneration. For standardizing the regeneration

protocol, the four wheat varieties, two each oiaesiivum and durum were utilized.

4.1.1. CALLUS INDUCTION

A wide range of variation was observed for callus induction when embryos of

different age groups (10-18 DPP) were cultured on following four callus induction

media Cl (MS+2,4-D, 2mg/i), C2 (MS+2,4-D, 4mg/l), C3 (MS+2,4-D, 2mg/l+lAA,

lmg/1), C4 (B5+2,4-D, 2mg/l). 25 to 35 embryos of each age group were cultured on

all the combinations of the media.

4.1.1.1. Effect of callus induction media

Mean values for callus induction frequencies in case of wheat varieties UP

2338, WH 542, Bijaga Yellow and Gulab were always significantly better on Cl

(83.00, 73.33, 78.18 and 78.00 percent) and C2 (83.33%, 87.35, 84.26 and 81.44

percent) in comparison to C3 (62.65, 57.60, 62.59 and 62.67 percent) and C4 (62.35,

55.60, 63.04 and 68.67 percent respectively). Differences between mean callus

induction on CI and C2 were non-significant (at 5% P) except for Bijaga Yellow

where it was significantly higher on C2 (Table 8). In general, 62.50-100% embryos on

Cl, 66.67-100% on C2, 37.93-76.67% on C3 and 46.15-83.33% on C4 transformed

into callus. All the embryos belonging to bread wheat varieties UP 2338 (12 DPP) and

WH 542 (12 and 14 DPP) transformed in to callus on C2 medium. 100 % callus were

also obtained from 12 days old embryo of WH 542 on Cl medium. Whereas, a

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Fig 3. Explanations

Different types of induced calli

A: Loose and white non embryogenic callus.

B: Same with improved texture callus mass.

C: Callus showing initiation of green sectors.

D: Typical organogenic callus with emergence of shoot primordia.

E: Typical embryogenic callus showing somatic embryos at different developmental

stages.

F: Embryo showing precocious germination and poor quality callus.

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Results 33

maximum of 97% and 90% callus induction was recorded in durum wheat varieties

Bijaga Yellow (12 DPP) and Gulab (12 and 14 DPP, respectively) on C2 medium

itself.

With regard to quality, calli on CI were glistening white yellow to pale

yellow, compact, opaque, nodular and embryogenic (preferable) (Fig. 3 E) whereas

those of C2 were transparent white, loose, watery and non-embryogenic (non-

preferable) (Fig. 3A & B) . The calli induced on C3 and C4 were loose to compact,

yellow-white, coarse, friable and non-embryogenic with occasional green sectors

(Fig. 3, C, D).

4.1.1.2. Influence of age of embryos (DPP)

Optimum age of embryos for induction of callus for both aestivum and durum

were found to be 12-14 and 12-16 DPP respectively (Table 8). No significant

difference (at 5% P) were observed for mean percent callus from 12 and 14 days old

embryos of variety UP 2338 (84.43 and 86.47%) and WH 542 (83.33 and 82.44%).

Differences among mean callus induction frequency of 12, 14 and 16 DPP embryos of

Bijaga Yellow (80.42, 83.33 and 81.55%) and Gulab (80.00, 82.50 and 76.80%) were

also non significant.

4.1.2. REGENERATION

Calli were transferred on different MS based mediums i.e. Rl (left on the

same medium and exposed to 16/8hrs light/dark, R2 (MS + Kinetin, 2mg/l), R3 (MS

+ Kinetin, 2mg/l + lAA, 0.5mg/l), R4 (MS + BAP, 2mg/l + lAA, 0.5mg/l), R5 (MS +

BAP, 2mg/l) media to get regeneration. Experiments were designed to evaluate the

effect of regeneration medium, callus initiation medium and age of explants on

regeneration frequency and multiple shoot formation (Fig. 4). Callus induction

medium was recorded to have very influential effect on regeneration especially when

exposed to light (Rl). As for all the four varieties tested, calli induced on CI

exhibited quite appreciating regeneration, while there was no response from calli

induced on C2. C3 and C4. Therefore, while performing statistical analysis Rl was

omitted from regeneration data on C2, C3and C4 induced callus.

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Rc'siihs 34

4.1.2.1. Bread wheat

Both callus induction as well as regeneration media had a significant influence

on regeneration frequency. Calli of UP 2338 induced on CI, C2, C3 and C4 reported

to give 30-90, 20-56, 21-68 and 29-63 percent respective regeneration, whereas

WH 542 exhibited 33-100, 20-56, 19-63 and 20-60 percent regeneration, respectively.

Maximum regeneration was observed from calli induced on CI medium.

Similarly on R2, R3, R4 and R5 media, 33-90, 23-63, 20-56 and 30-92 percent

calli of UP 2338 and 33-100, 18-57, 20-67 and 30-90 percent calli of WH 542

exhibited multiple shoot formation, respectively. In both the varieties mean frequency

of regeneration on R2 and R5 were significantly higher than R3 and R4, irrespective

of the callus initiation medium or age (Table 9). Plantlets on R3 and R4 were also thin

fibrous and delicate in comparison to that of R2 and R5. Albino plants were recorded

from calli of WH 542 induced on C2 and regenerated on R3 medium. Regeneration

on Rl for both UP 2338 and WH 542 were good from calli induced on CI. ( ^<Q-^)

Age of embryos also exerted a significant influence (at 5% level) on

regeneration frequency. In both the varieties, 10 DPP embryos gave maximum

regeneration among all the treatment groups. Frequency of regeneration was found to

decrease with increasing age of embryos.

4.1.2.2. Durum wheat

Callus induction and regeneration medium, as well as age of the embryos, all

exerted a statistically significant influence on regeneration. Calli induced on CI, C2,

C3 and C4 medium exhibited a respective regeneration frequency of 25-91, 20-63,

14-50 and 14-67 percent from Bijaga Yellow and 27-92, 11-55, 14-50 and 17-63

percent from Gulab. Maximum regeneration was always recorded on CI medium

(Table 10).

Regeneration on Rl was recorded to be 61-91 and 75-92 percent from Bijaga

Yellow and Gulab from calli induced on CI, whereas calli on the C2, C3 and C4 gave

negligible regeneration on exposing to light. A regeneration frequency of 33-88,

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Page 61: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Fig 4. Explanations

Moqjhogenesis in different types of calli

A: Organogenic calhis with poor regeneration efficiency.

B: Callus showing regeneration pockets.

C&D: Regeneration from embryogenic calli (arrow showing germinating embryoids).

E: Organogenic callus (showing shoot as well as root formation).

F: Calli exhibiting both somatic embryoids (arrow) and shoot buds (green colored).

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Results 37

14-63^ 14-54 and 20-86 percent from Bijaga Yellow and 25-86, 11-57. 13-43 and 20-

83 percent from Gulab were gained on R2, R3, R4 and R5 respectively.

10 DPP embryos gave significantly better regeneration from CI derived calli.

However, 12 DPP embryos were also better responding on other media compositions.

Embryos were found to be loosing their regeneration potential along with their

growing age.

4.1.3. INTERACTION OF CALLUS INITIATION AND REGENERATION MEDIUM

To study the interaction of callus initiation and regeneration medium, a

hypothetical regeneration per 10 explants cultured was calculated as,

% callus induction x % regeneration (•/,) 10

Both regeneration frequency and regeneration per 10 explants was plotted for

the same DPP embryos and analyzed. By computing this regeneration per 10 explants,

effective regeneration can also be comprehended.

4. L3.1. Bread wheat

Callus induction frequency from immature embryos of UP 2338 was quite

high on both CI and C2 media. Mean callus initiation on C2 was higher in

comparison to CI. However, these calli when transferred to regeneration medium, the

calli induced on CI tend to produce multiple shoots more frequently in comparison to

that of C2. This ultimately affected the effective regeneration which was always high

from calli of CI (Table 11). A constant maximum regeneration was attained on both

R2 and R5 as well.

Mean frequency of callus initiation for WH 542 was higher on C2 in

comparison to CI, C3 or C4 and mean regeneration was maximum from calli induced

on CI (Tables 9 and 10). Though the frequency of callus initiation was higher on C2,

the calli were watery, loose and non-embryogenic. When such type of calli were

transferred to regeneration medium, root induction preceded the shoot initiation and in

most of the calli no shoot was formed at all. Regeneration per 10 explants cultured

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Fig 5. Explanations

Different development stages

A: Embryogenic callus of varityGulab.

B,C & D; Multiple shoot proliferation from subcultured calli

E; Emergence of roots from green sectors of watery calli.

F,G & H: Developmental stages of regenerated plantlets.

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Results 39

revealed that effective regeneration was always higher from CI induced calli than the

calli from C2, C3 and C4 (Table 11). Multiple shoot formation on both R2 and R5

was equally good and showed maximum regeneration.

4.1.3.2. Durum wheat

By analyzing the callus induction and regeneration responses of embryos of

Bijaga Yellow (Tables 8 and 10), it was apparent that though maximum callus

induction could be obtained on C2, maximum and effective regeneration

(regeneration per 10 explants cultured) was higher from the callus induced on CI

(Table 12). R2 and R5 were equally responding for both regeneration frequencies as

well as induction of multiple shoots.(Fig-5)

Frequency of callus induction from embryos of Gulab was higher on C2,

whereas calli induced on CI showed maximum regeneration. Effective regeneration

was also significantly higher from calli induced on C1. Both R2 and R5 gave a good

number of induced multiple shoots.

4.1.3.3. Statistical inference

For all the four varieties tested, effective regeneration was always maximum

from the calli induce on CI in every age group. Regeneration on Rl was quite

appreciating from calli on CI medium (Tables-11 and 12). R2 as well as R5

responded well irrespective of callus induction medium. Regeneration from R3 and

R4 were ought to be very poor.

4.1.4. SHOOT PROLIFERATION AND PLANTLET ELONGATION

For further proliferation of regenerated calli three different pathways were

deployed: SI, (left on the regeneration medium itself), S2, (MS + GAs 0.5mg/l +30g/l

sucrose) and S3 on (GR free MS). Number of plantlets per callus was recorded

irrespective of the age of embryos. Only calli induced on CI were subjected to study

for this parameter. Mean values of shoots per callus were calculated from five calli

and depicted in Table 13.

4.1.4.1. Bread wheat

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Page 70: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 41

Regenerated calli of UP 2338, when transferred to S3, got established

maximum number of shoots per callus. However, when left on the same medium few

plantlets got dried, thereby reducing the number of plantlets. Regenerants when

transferred on S2, turned brown and only a few healthy plants were able to survive.

Maximum plantlets (20.4) were obtained from calli regenerated on R2 and later on

transferred to S3. Number of plantlets per 10 explants cultured led to obtain the

maximum plantlets on transferring to S3 from calli regenerated on R2 (140.56),

followed by R5 (125.94) and Rl (115.37).

Responses of WH 542 to shoot proliferation medium were in accordance to

UP 2338. Maximum plantlets (26.0) were obtained on S3 from calli regenerated on

R2. Maximum number of plantlets per 10 embryos were achieved on S3 itself from

the calli regenerated on R2 (188.76) followed by R5 (136.60) and Rl (132.00).

However, maximum regenerants were obtained on Rl medium (Table 13).

4.1.4.2. Durum wheat

Variety Bijaga Yellow was found to give maximum shoots per callus on S3

medium from calli induced on R5 (16.8) followed by R2 (15,4) and Rl (14.6).

However, mean values for plantlets per 10 explants were maximum on Rl (66.70)

followed by R5 (61.92) and R2 (56.78).

Although maximum regeneration from variety Gulab was obtained on Rl,

mean values for both shoots per callus and number of plantlets per 10 embryos were

found to be maximum from calli induced on R5 (Table 13).

4.1.4.3. Statistical inference

Statistically, S3 was found to give maximum number of shoots per callus and

differences among SI, S2 and S3 were found to be significant.

4.1.5. RHIZOGENESIS IN REGENERANTS

Healthy, white adventitious roots are prerequisites before going for hardening

and acclimatization of plantlets regenerated in vitro. During the present study it was

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Results 43

observed that among all the varieties tested, roots were also induced along with shoots

on regeneration medium itself, but quality was very poor. Such type of plantlets

failed to survive the hardening and acclimatization process. Therefore, to achieve a

good rooting plantlets with 2 to 3 leaves were transferred on a range of medium

having different growth hormones, sucrose content and with or without added

vitamins (Table 14). Most of the roots induced on Rt 1, 2, 3 and 5 were thin, fibrous

green and could attain a length of about 8-10 cm with lesser lateral branches. These

were therefore not suitable to go for plant hardening. However, roots emerged on Rt 4

medium were white, healthier and thick with lateral branches. These were considered

to be suitable as responded suitably during hardening and acclimatization of plantlets.

4.1.5.1. Inference

On the basis of the experiment conducted during investigation by examining

the role played by the medium having MS salts (without vitamins) + NAA (1 mg/1) +

Sucrose (20 g/1), without adding vitamins was found to be ideal for root induction and

subsequent hardening of the plantlets.

4.1.6. HARDENING AND ACCLIMATIZATION

Three different pathways were adopted for hardening and acclimatization of

plantlets (Chapter 3, Materials and methods). The success rate of established plants

was only 10 percent when plantlets were hardened using the first method about 20-30

percent plantlets survived in the second method. Decrease in success through these

methods may be attributed due to contamination during several sub-culturing. Only a

few plants withstood the shift from solid to liquid and then soil mixture route.

Nearly 50 to 90 per cent plants survived when hardened and acclimatized

through third method. This condition to success was having good root system and

stable low temperature and high humidity. Also plants did not have to withstand

several sub-culturing shocks passing through semi solid, liquid and soil phases.

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Page 74: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Fig 6. Explanations

Hardening and acclimatization of plantlets

A: Plantlets in liquid medium.

B: Semi-hardened plant in growth chamber.

C«&D: Hardened plants in field.

E&F: Established Ro plant of UP 2338.

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Page 76: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 45

EXPERIMENT-2

4.2. SELECTION OF THE BETTER RESPONDING GENOTYPES

Adopting the abovementioned protocol, eight varieties of each, bread and

durum wheat varieties were screened for their ability to form callus and to explore the

regenerative potentiality of the calli.

4.2.1. CALLUSING

Among the 8 wheat varieties tested, cent per cent callus induction was

exhibited by 12 DPP embryos of WH 542. No marked difference was observed

among UP 2338, PBW 343, PBW 373, Raj 3765, HD 2329 and WH 542 for their

mean callus induction frequencies (Table 15). Comparatively poor callusing was

recorded from CPAN 3004 and KRL 1-4. Embryos aged 12, 14 or 16 DPP exhibited

higher callusing depending on the genotype.

In general, the durum wheat embryos were found to have lesser tendency to

transform into callus as compared to bread wheat. A maximum of 87 per cent

embryos of Bijaga Yellow and Gulab exhibited callusing to bread wheat. Mean

values for callus induction were higher in embryos taken from Bijaga Yellow, PDW

215 and Gulab (Table 15), followed by PDW 233 and Raj 1555 (both fall in same

significance level). Durum varieties HI 8381, MACS 2846 and HD 4530 responded

very poorly to callus initiation. Significantly better callusing was obtained from 12 to

18 DPP embryos depending on the genotype.

4.2.2. PRECOCIOUS GERMINATION

In the present study, older embryos (16 and 18 DPP) exhibited a tendency to

germinate precociously i.e. without attaining maturity. Maximum precocious

germination among bread wheat was recorded in varieties showing minimum callus

induction i.e. in KRL 1-4 (26%) and CPAN 3004 (20%) from 18 DPP embryos

followed by HD 2329, Raj 3765 and PBW 373. Minimum precocious germination

was observed in UP 2338 and WH 542 (Table 15).

Among durum wheat, HI 8381 and MACS 2846 showed maximum precocious

germination. Even 14 DPP embryo of HI 8381 germinated on callus initiation

Page 77: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 78: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 47

medium. Embryos of Bijaga Yellow, Raj 1555 and Gulab showed lesser tendency to

germinate than PDW 215 and PDW 233.

4.2.3. REGENERATION

Ri R2 and R5 pathways were adopted to test the regeneration ability of the

cultivars.

4.2.3.1. Regeneration on Rl medium

Among bread wheat varieties cent per cent regeneration was obtained in case

of WH 542. PBW 343 and HD 2329. However, mean values for regeneration on all

the five days were significantly higher for WH 542 (92%) at 5% level of significance

(Table 16). Regeneration from UP 2338, PBW 343, PBW 373, Raj 3765 and HD

2329 was moderately higher and did not differ much from each other. CPAN 3004

and KRL 1-4 revealed poor regeneration.

Regeneration in durum wheat varieties Bijaga Yellow, Raj 1555, PDW 215,

PDW 233 and Gulab was quite high and their mean values did not show much

differences (Table 16). However, the response of HD 4530, HI 8381 and MACS

2846 was found to be poor. In general, age of embryo exerted a significant influence

on the regeneration frequency as the 10 DPP embryos always exhibited maximum

regeneration.

4.2.3.2. Regeneration on R2 medium

Among bread wheat varieties, on R2 medium, 100 per cent regeneration was

obtained in case of WH 542 and PBW 343 (Table 17). Response of UP 2338, PBW

343 and PBW 373 also got the same place as they fell in the same significant level

followed by PBW 373, Raj 3765 and HD 2329. Varieties CPAN 3004 and KRL 1-4

responded very poorly.

Durum wheat cultivars, Bijaga yellow, Gulab and Raj 1555 were found to be

ideal responding genotypes followed by PDW 215 and PDW 233 (Table 17). Rests

were poor in regeneration.

4.2.3.3. Regeneration on R5 medium

Bread wheat varieties UP 2338 and WH 542 were ideal on R5 medium

followed by PBW 343 and PBW 373 and subsequently by Raj 3765 and HD 2329

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Page 81: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 50

(Table 18). CPAN 3004 and KRL 1-4 were very poor in their response to this

medium.

Durum Wheat varieties PDW 233 and Gulab were found to be better followed

by Bijaga Yellow, Raj 1555 and PDW 233. Raj 1555 and PDW 215 were next in

their regeneration response. Rests did not show any encouraging response.

4,2.3.4. Selection of the varieties

On the basis of the present findings, among bread wheat varieties, WH 542

ranked the best for its regeneration potential followed by PBW 343 and UP 2338

(Table 19). However, these three varieties stood at the same level of significance

ladder (at 5%P). PBW 373, Raj 3765 and HD 2329 were found to be moderate in

their ability to regenerate. Performance of durum wheat varieties Gulab and Bijaga

Yellow was quite appreciating followed by Raj 1555 and PDW 233. Performance of

PBW 215 was also good.

Therefore, depending on their ability to regenerate following varieties of bread

and durum wheats were selected to be used in further crossing program.

Bread wheat Durum wheat

WH542

UP 2338

PBW 343

Raj 3765

HD 2329

Raj 1555

Bijaga yellow

Gulab

PDW 215

PDW 233

Page 82: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 84: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 53

EXPERIMENT-3

4.3. EMBRYO RESCUE AND WIDE CROSSING

Five varieties each of bread and durum wheats were crossed with different

Triticum and Aegilops species (Table 20). Embryos were rescued at 10, 12, 14 and

16 days post pollination. Calli were induced on CI, MS + 2mg/l, 2,4-D and

regenerated on R2, MS + 2mg/l Kinetin or R5 2 mg 1'' BAP + 0.5 mg/1 lAA media .

All the three media were standardized in the laboratory.

In hybrids involving T. urartu as one of the parents, 14 DPP embryos of PBW

343 exhibited maximum callusing (48.48%) and 10 DPP of HD 2329 gave the least

(16.67%) among bread wheat varieties (Table 21). Moreover, these two combinations

again responded to maximum and minimum regeneration (66.67 and 46.88%,

respectively). In durums, the varieties Gulab (14 DPP) and Raj 1555 (10 DPP) with

urartu showed the highest (44%)) and the least (12.50%)) callusing, respectively.

However, there was not very apparent difference in crosses for regeneration. Much

older embryos (>16 DPP) of crosses with WH 542, UP 2338, Gulab, Raj 1555 and

PDW 233 were distorted in shap^F .' Embryos of Raj 1555 x T. urartu were so

vigorous in inducing callus that it required 30 to 40 vessels to maintain calli induced

from a single embryo. Plants established in the field were tall and long spiked but

with poor seed setting.(Fi^ 9)

Embryos of hybrids with T. boeticum were rescued at different DPPs viz.,

with WH 542 (10-16 DPP), PBW 343 (10-14 DPP) and Bijaga yellow and PDW 215

(9 to 15 DPP) (Table 21). The callus induction frequency in crosses involving bread

wheat varieties was recorded highest (47.06%)) and the minimum of 11.76 per cent

from 12 and 16 DPP in hybrids with WH 542 giving 42.86%) regeneration. The

variety PBW 343 carrying IB/IR translocation was found comparatively less

responding to callus induction and regeneration. Among crosses with durum, 14.29%)

(from 10 DPP) to 28.57% (12 DPP) calli of hybrid with Bijaga Yellow exhibited

callusing which either became brown or turned into roots. On the other hand,

embryos from the hybrids involving PDW 215 did not respond to callus induction

medium.

Embryos of hybrids with Ae. speltoides were scooped at 10 to 16 days from

hybrids with WH 542, 10 to 14 days PBW 343, 9 to 15 days UP 2338 and HD 2329

Page 85: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 86: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 55

and 8 to 14 days post pollination from durums (Table 21). Success to callus induction

was found to 9.09 (lODPP, WH 542) to 30.77 per cent (13 DPP, PBW 343) and nill

regeneration (HD 2329) between 28.57% (UP 2338). Only the embryos of 10 and 12

DPP in durums responded to callus induction from 16.67 to 40.00%, with PDW 233

only regenerating genotype (25.00%). However, callus could b&«i)tdi?ied ^gpri 8 and

14 DPP embryos. Plants of the crosses with UP 2338 a^JPDW 233 got es tabh^d II ( /i..., -v-, ) \

in field with poor seed setting. ' > ^ _ ,' Sjj

Hybrid embryos of crosses with Ae. squarrosa werevjirj^ued either^ 10 to

14 DPP (with Gulab and Raj 1555), 10 to 16 DPP (with WH 542, UP''233'8; PDW 215

and PDW 233) or 10 to 17 DPP (HD 2329 and Raj 3765). Frequency of callus

induction ranged from no callusing (17 DPP. Raj 3765) to 60.00% (12 DPP, WH 542)

among aestivums and nil (10 and 14 DPP Gulab) to 54.55% (12 DPP, PDW 233)

among durums (Table 21). No marked difference was observed for regeneration.

However, no multiple shoots could be regenerated from calli of the crosses with Raj

3765 and Gulab. HD 2329 possessed distorted seed at 17 DPP. Hybrids involving

HD 2329, Raj 1555 and PDW 233 were raised to maturity with poor seed settingCFf^a).

Among the hybrids involving Ae. markgrafii, all embryos were rescued at

10, 16 DPP and in vitro responses of hybrids with only one durum variety Raj 1555

was found better (38.89 to 61.11%) callus induction and 63.64% regeneration) than

other with bread wheats showing 33.33 to 56.56%) callus induction and 56.76%)

regeneration from HD 2329 and 37.14 to 55.00 % callus induction and 50.00%)

regeneration from UP 2338 (Table 21). Plantlets of the cross with UP 2338 and Raj

1555 were grown to maturity but without any seed settingCFi'g lo).

Hybrid embryos with Ae. umbellulata were rescued at different days post

pollination i.e. at 10 to 14 DPP with Raj 1555 and PDW 233, 10 to 16 DPP with PBW

343 and 12 to 18 DPP with WH 542. No marked differences were observed among

hybrids of two varieties of bread and durum wheats, as both responded quite

vigorously to callus induction (35.00 to 62.50 and 25.00 to 53.85%) and regeneration

(53.33 to 62.07 and 54.07 to 55.00 % respectively) (Table 21). Hybrids involving Raj no

1555 and PDW 233 got established in field, however,feeed setting was observed.

Embryos of crosses involving Ae. cylindrica were rescued at different days

post pollinations viz., 9 to 15 DPP from crosses with Raj 3765; 10 to 15 with PBW

343; 10 to 16 with UP 2338 and HD 2329; 10 to 14 with Bijaga Yellow, Gulab and

Raj 1555; and 8 to 14 with PDW 215 and PDW 233. Frequency of callus induction

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Re.sii/l.s 56

was quite good from embryos of hybrids with bread wheat (30 to 47.37%).

Regeneration was very poor with 20% from WH 542 and 24% from UP 2338. CalH

from other crosses could not be differentiated into shoots. 10 to 14 DPP embryos of

durum hybrids turned into callus (10.00 to 25.00%), while 8 DPP embryos didn't

responded (Table 21). All the calli turned into roots only. Some of the embryos of all

age groups of Gulab and 14 DPP of PDW 215 and PDW 233 got abnormal

protuberances. Hybrid HD 2329 x Ae. cylindrica attained maturity with poor seed

setting.

10 to 16 DPP embryos of the hybrids with Ae. ventricosa were cultured.

Only hybrids with WH 542 transformed into callus (10.00 to 25.00%) and regenerated

as well (33.33%)) (Table 21). No calli could be obtained from the hybrids with PBW

343 and Bijaga Yellow. Hybrid plants involving WH 542 could be raised but

exhibited poor seed setting.

In crosses with Ae. kotschyi 10 to 16 DPP embryos of the hybrids with single

bread wheat, PBW 343 and 9 to 15 DPP of durum were cultured. Although, 12.50 to

30.00% callus was obtained from PBW 343, however, none of the calli could turn into

shoots. The durum wheat hybrids exhibited better callusing (16.67 to 57.14% on 10

and 12 DPP, respectively) and even 52.63%) calli of Raj 1555 and 52.17% calli of

PDW 215 transformed into multiple shoots (Table 21). The calli of hybrid with

Gulab also turned brown producing roots only. The hybrids involving Raj 1555 and

PDW 215 were raised to maturity with very poor seed settingCKa \o\

Hybrid embryos with Ae. peregrina were rescued at 10 to 16 DPP . Among

bread wheat varieties, Raj 3765 was found better with maximum callus induction

(37.71%) froml2 DPP) and regeneration (44.44%)). Among durums. Raj 1555 was

found better with maximum callus induction (46.67%) from 14 DPP) and maximum

regeneration (47.37%) (Table 21). The hybrids with Raj 3765, Raj 1555 and PDW

233 got established with very poor or no seed setting.

10 to 18 DPP embryos of the hybrids involving y4c. triuncialis with bread

wheat and 9 to 16 DPP of durum were cultured with 22.22 to 64.29%) and 20.00 to

45.45%) success (Table 21). The young hybrid embryos of durums (9 and 10 DPP)

did not respond, however, all the calli became brown and no further development

could be achieved.

Page 88: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 90: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 91: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Results 60

Hybrid embryos of Ae. geniculata as one of the parents were scooped at 10

to 16 days post pollination and cultured successfully. Among the crosses with bread

wheat, Raj 3765 was found better responsive for both induction of calli (a maximum

of 53.33% from 12 and 14 DPP embryo) and regeneration (60.87%). The frequency

of callus induction was recorded highest from the crosses with Gulab (54.55% from

12 and 14 DPP) and regeneration from Bijaga Yellow (52.63%) (Table 21). The

plantlets of the cross with Bijaga Yellow got established in field with no seed setting.

Hybrids involving/4e. ovata were also rescued at 10 to 16 DPP. The bread

wheat hybrid with WH 542 exhibited highest (63.75% at 14 DPP) calli formation and

better regeneration (62.88%) in comparison to hybrids with UP 2338. Among durums

the maximum (53.85%, 12 DPP) and minimum (37.50%, 10 DPP) callus induction

was attained in the hybrid embryos with Bijaga Yellow, whereas maximum

regeneration (60.87%) was recorded from PDW 233 (Table 21). Both callus induction

and regeneration were found good, inspite of having abnormal embryos (in shape)

from WH 542, Bijaga Yellow and PDW 233. The hybrid plants with WH 542, got

established in the field, with spikes having open florets at the time of pollination

(Fig. 8). However, very very poor seed setting was recorded.

Hybrids embryos with T. araraticum were rescued at 10 to 16 DPP. The

hybrid embryos with bread wheat variety UP 2338 did not respond to callus induction.

Whereas, 20.00 to 68.33% embryos of the hybrids with durum turned into callus

(Table 21). The organogenesis from all these hybrid embryos ended with the

formation of roots only.

None of the crosses with Ae. longissima and Ae. sharonensis (Table 22)

possessed dissectible embryos as the caryopsis either became white and epaque with

no content inside or green with watery fluid inside. Embryos of hybrids with Ae.

bicornis, Ae. caudatei Ae. lorentii and Ae. triaristata taken as male parent (Table 23)

were rescued at different days post pollination ranging from 8 to 18 days. However,

none of the embryos transformed into calli.

Page 92: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

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Page 93: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Fig 7. Explanations

Normal and abnormal embryos rescued from wide crosses

A: 8 days old embryo of bread wheat.

B: 18 days old normal embryo.

C: 14 days old Gulab x T. urartu hybrid embryo (abnormally small size).

D: Hybrid embryo of UP 2338 x Ae. bicornis (trilobed).

E: Hybrid embryo of WH 542 x Ae. ovata (abnormal protuberance).

F: Hybrid embryo of Raj 1555 x Ae. peregrina (shapeless).

G: Hybrid embryo of PDW 233 x Ae. triuncialis (no distinct apical axis).

H: Hybrid embryo of PBW 2\5xAe. cylindrica ( twin embryos).

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Fig 8. Explanations

Regeneration from embryos along with peculiar hybrid spikes.

A: UP233S\Ae. cylindrica.

B: PBW 343 X r. wrar/w.

C; In vitro raised branched spike from UP 2338 x T. urartii.

D. WH 542 X Ae. speltoides.

E: PDW 233 xAe. squarrosa

F: Hybrid spike at WH 542 x Ae. ovata showing open floret at the time of

pollination.

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Fig 9. Explanations

AB&C: Regenerants induced via callus cultures of single embryo hybrid of

Raj 1555 X r. urartu.

D: Hybrid plants of PDW 233 x T. urartu.

E: Fi ears of Raj 1555 x T. urartu.

F: F2 ears of hybrid of Raj 1555 x T. urartu with female (left) and male

(right) parents.

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Fig. 10 Explanations

Different wild hybrid plants established in field through embryo rescue

A: WH 542 xAe. ovata.

B: Raj 1555 x 7". iirartu.

C: UP 2338 xy t;. markgrafii

D: Bijaga Yellow x T. urartu

E; Raj \^S^xAe. kotschyi

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VtSCUSSZON

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5. Discussion

To keep pace with the global wheat demand, there is a need to break the

existing yield plateau, stabilize production at each level of production ladder, and

extend wheat cultivation to problem and marginal areas and breed wheat varieties

with specific industrial and nutritional requirements. This ultimately necessitates

broadening the genetic base through alien gene transfer and one alternative to

accomplish this in wide hybridisation. Literature reveals that agronomically useful

hvhrids, even between closely related species, are often unattainable due to sexual

incompatibility. In many instances fertilization however takes place, but the

de\ eloping embryo aborts at various stages of its development. The techniques of

embryo rescue which involves the excision and culture of the young hybrid

embryos to obtain hybrid plants can play vital role in creating variability and

pyramiding the useful genes (Collins and Grosser, 1984; Raghvan, 1986).

During the present course of study, before gomg straw for rescuing embryo,

it was tried to develop a suitable regeneration protocol and select out the better

responding genotype in vitro. Although, several other factors can affect the callus

induction and plant regeneration from rescued embryo. If we have a better

responding genotype as one of the parent, the chances of getting regenerants

become high (Ou et al, 1984). An established protocol can also help in going for

transformational studies through various methods (Becker et al, 1994; Karen et

al. 2000 and Huber et al, 2002) including somaclonal variation (Villareal et al,

1999; Benkirane et al, 2000), screening of genotypes against various stresses (Jia

and Zhao 1993; Wang et al, 1993) or in vitro mutational studies (Liu et al, 1993).

5.1. BASAL CULTURE MEDIA

Two types of basal culture media MS and B5 with same GR supplemented

i.e.. 2mg/l 2,4-D were used. Calli raised on MS were found to be embryogenic

and more regenerative, whereas, that on B5 were organogenic and have lesser

regeneration frequency. Both the media MS and B5 differe in nitrogen (NH4NO3

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Discussion 63

& KNO3) and phosphorous source (KH2PO4 and NaH2P04), respectively. The

amount of CaCl2 , MgS04, KI, H3BO3, MnSo4, ZnS04 are also different. Any of

these compound(s) may have a. influence on role of culture responses and may lead

to poor or good regeneration. He et al. (1991) also noted that the ingredients of

basal culture media and their amount influence the in vitro responses. Nitrogen

source has been reported to affect the embryogenesis by Higashi et a/. (1996).

5.2. GROWTH REGULATOR

In the present course of study it was observed that growth hormones have a

vital role to play at each step to get regeneration. On the MS medium containing

2mg/l 2,4-D typical embryogenic calli were obtained characterized by glistening

pale yellow/yellow white surface, compact and opaque, which latter on became

nodular with buds. Embryogenesis from similar type of calli were also reported

by Ozias-Akins and Vasil, (1983a,b); Immonen, 1996; Farooq et al, 2001; Heyser

el ai, 1985; He et al, 1986 and Hunsinger and Schauz, 1987. Embryos transferred

on media containing 4mg/l 2,4-D were very vigrous, however, calli were loose

watery, fragile and transparent, more likely to transform into roots (Chauhan and

Singh, 1995). However, when immature embryos were planted on growth

hormone free MS media no callusing was observed. Addition of auxins were

found necessary to induce callus, synthetic auxins were supposed to disorient the

previously programmed patterns of cellular activity (Wernicke and Milkovits,

1984) and permit or stimulate cellular organization and embryoid formation in

some unknown way.

Poor callusing and regenerability at higher 2, 4-D concentration may be an

artifact of quantifying regeneration without allowing adequate time for

regeneration of the less mature embryos (Bregitzer et al, 1995). By using an

anti-auxin TIB A into medium Cistue et al (1999) concluded that by reducing

auxin, calli could be obtained from otherwise poor responding anthers, however

2,4-D was essential to elicit the response.

2, 4-D in combination with lAA leads to the formation of fragile calli with

green sectors, which latter on give rise to plant organogenically. Shoots were also

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Discussion 64

reported to originate from such green sectors by Shimada 1978; Ivanov et al., 1998

and Machii et al., 1998. 2 mg/1 2,4-D was found to be optimum and supported by

Sear and Deckard et al., 1982; 1983; Mathias et al., 1986 and Ozyen et al., 1998.

No embryoids were recorded in calli induced on MS + 2,4D (4 mg/1), MS +

2,4D(4 mg/1) + lAA (1 mg/1) and B5 + 2,4-D (2 mg/1). This support the view that

embryogenesis in wheat callus culture was affected by phytohormone

concentration and type (He et al., 1986; Hunsinger and Schauz, 1987; Redway,

1990). In present study it seems that lAA inhibits the callus induction and also

deteriorates the quality. No callusing was observed on medium containing lAA.

This was also supported the view of Ozias-Akins and Vasil (1982). Embryogenesis

was preferred over organogenesis, as single cell origin of former leads to

genetically uniform plantlets, instead of chimeric ones (Haccius, 1978; Vasil and

Vasil, 1982; Vasil, 1987).

Addition of cytokinins like Kinetin and BAP stimulates shoot formation in

callus (Shrivastava el al., 2000). 2 mg/1 of kinetin was found to improve shoot

regeneration (Bosch Wackerle et al., 1979; Papenfuss and Carman, 1987),

however, in combination to 0.5 mg/1 lAA leads to rooting instead of shoots. On the

other hand 2 mg/1 BAP in combination to 0.5 mg/1 lAA gave better shoot

formation than BAP alone and lead to conclude the interactive effects of hormone.

BAP was also used by Hussain and Islam (1993) and Mathias et al. (1982) to get

regeneration. It was observed that callus induction media also have a profound

effect on regeneration ability of the calli in association to that of regeneration

media. It was in accordance to Ward and Jordan (2001), who postulated that

composition of callus induction media affects both, the percentage of callus

formation from embryos and subsequent frequencies of plant regeneration.

However, they found that regeneration medium had no effect on level of plant

regeneration, instead during our experimentation it was recorded that regeneration

frequency was also very much affected by composition of the regeneration

medium. R2 and R4 were found to give statistically better response than R3 and

R4.

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Discussion 65

Shoot enlarged on GR free media with reduced sugar (20 mg/1) produced

maximum number of multiple shoots in comparison to that left on the same

medium on which they were induced. The reason can be, firstly the mechanical

injury faced by grown up plantlets with the 3-4 leaves during taking out from the

flasks; and secondly, additional nutrition provided by fresh subculture leads to the

development of each and every plantlet thereby increasing the number of

plantlets/callus. As plantlets established their own endogenously supplied growth

hormones, there was found no need to supply it artificially. Plantlets grown on

reduced sugar (S3 and Rt4) were having healthier shoots and roots. Reducing

sugar from the medium may be beneficial to plantlets by promoting

photoautotrophy in vitro and also by reducing the loss of plantlets due to

biological contamination of culture medium (Kozai, 1991, Seko and Nishimura,

1996).

During present course of study, addition of GA3 (0.5 mg/1) to the medium

caused browning at the base of the plantlets. This deteriorates the number of

shoots and even most of the plantlets get dried within 3 to 4 weeks. However, use

of GA3 was recommended to induce root formation (Ozias-Akins and Vasil, 1982)

and in regeneration medium (Bohorova et al, 1995). Callus induction rate,

regeneration frequency, number of plantlets regenerated were all found

independent of each other (Ozygen et al. 1998). Albino plant was recorded in

WH 542 from calli induced on C2 and regenerated on R3. The reason may be high

concentration of 2, 4-D (4 mg/l)in comparison to that of other initiation media (2

mg/1) and the cultivar may be more sensitive to changes.

5.3. PRECOCIOUS GERMINATION

Precocious germination is the ability of embryos to germinate without

attaining maturity. The phenomenon of precocious germination is not desirable as

it results in smaller and less vigorous seeds and poor quality of calli (Ozias Akins

and Vasil, 1982). In present course of study a very few zygotic embryos were

found to germinate precociously. Smaller embryos of 10, 12, 14 or even 16 DPP

do not germinate except durum wheat HD 4530 which germinate (3.33%) at 14,

DPP even. Culture of embryos by planting them with scutellum side upward, was

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Discussion 56

used as a strategy to check the germination of embryos followed by Fenell et al. in

1996. Even than, some of the embryos of 16 and 18 DPP germinated. Minimum

germination was reported on medium supplemented with 4mg/l, 2,4-D than 2 mg/1

2,4-D. Media having lAA exhibited maximum germination. Several other

workers find decrease precocious germination with increased concentration of 2,

4-D (Ozias-Akins and Vasil. 1983; Carman et al., 1987). Papenfuss and Carman

(1987) used dicamba to suppress precocious germination with cent percent

success. However, calli and on dicamba containing medium were found necrotic

and even plantlets difficult to establish (Carman et al. 1987a). Distinct variations

were also observai among genotypes.

Carman et al. (1988) considered precocious germination of embryos as a

useful parameter for assessing the effect of callus medium on somatic

embryogenesis. Whereas, Immonen (1996) found it to be a cross specific/genotype

specific and therefore do not reflect the quality of callus induction medium. Bapat

el al. (1988) also treated the precocious germination of zygotic embryos as an

important characters, stating that genotypes that hui/e precocious germination of

zygotic embryos have greater embryogenic potential. However, findings of the

present study reflect a vice versa result.

5.4. AGE OF EMBRYOS

12 to 14 DPP embryos of aestivum wheat and 12 to 16 DPP of durum

wheat exhibited maximum callusing. Embryos less than 12 days were less likely

to transform into callus, however, older embryos (16 and 18 DPP) germinate

precociously. The similar trend was noted by Ward and Jorden (2001) in triticale.

They postulated that it is necessary for immature embryos to reach certain

physiological level to respond in vitro. The reason to this may be that 10 DPP

embryos were very small and delicate and get punctured/damaged during

dissection. 10 to 15 DPP embryos were reported to give fast and regenerable calli

by Shimada (1978), Magnussan and Bornman (1985) and Ozygen et al. (1998). 10

to 14 DPP was known to be optimum by Wang et al. (1993) and Arun et al.

(1994). Poor quality, lesser ability to regenerate and more tendency of having

precocious germination from older embryos can be attributed to the endogenous

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Discussion 67

supply of the growth hormones from them. As they can hinder the growth of calli

into callus and influence latter development in one or the other way.

Age of embryos also exert an influence in multiple shoot production from

calli. Maximum regeneration was attained from 10 days old embryo in almost all

the genotypes. This was in contrary to findings of Ward and Jordan (2001), as

they reported that 10 DPP embryos have poor ability to regenerate when compared

with others. Older calli (18 DPP) exhibited minimum regeneration with weaker

plantlets and poor rooting as also reported by Redway et al. (1990). Whereas^He el

ul. (1988) reported increased frequency of precocious germination in older

embryos. These characters were also found to be genotype specific as even 14

DPP embryo of durum wheat cultivar HDj was reported to germinate precociously.

5.5. GROWTH CONDITION OF CULTURES

Following Mathias et al. (1986) and Carman et al. (1988) cultures were

maintained in dark to induce callus at 25 ± 2°C temperature. Higher temperature

(30°C) leads to the browning of callus and excessive root formation.

Light during the regeneration phase were recorded to have a marked effect

as the calli induced on 2 mg/1 2,4-D when exposed to light provided by bulb

(20wt) lead to multiple regeneration sometime in 100% calli, on the same medium

in which they were induced. The reason could be lowering of auxin concentration

near the callus mass leads to the germination of embryoids precociously (Bapat et

al, 1988), as no regeneration were observed from non-embryogenic calli initiated

on C2, C3 or C4. However, when callus were exposed to light provided by cool

florescent tubes no regeneration was obtained. Type of light might also have some

role to play in addition of type of calli. Arun et al. (1994), also reported

germination of embryos on callus induction medium itself.

Light induced precocious germination of somatic embryos were also

reported by Ozias-Akins and Vasil (1983c) and Papenfuss and Carman (1987).

Where as regeneration on 2,4-D free medium was reported by Zhang (1986),

Taghvaii et al. (1998) and Shrivastava et al. (2000), however, the type of light was

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Discussion gg

not specified. Seko and Nishimura (1996) hypothesized that light is needed to

regulate morphogenic process of in vitro plantlets including shoot proliferation

and plantlet elongation,

5.6. GENOTYPE

In vitro responses were known to be genotype specific (Lang et al.. 1995;

Bankirane et al, 2000) in cereals along with wheat. Efficiency of callus induction

(Mathias and Simpson, 1986; Barkat and Abd-el-latif, 1995; Arzani and

Mirodjagh, 1999); Callus growth rate (Lazer et al., 1983), embryogenesis (Arun et

al.. 1994; J'Aiti et al., 1999) and plantlet regeneration (Karadimova et al.. 1985;

Machii et al.. 1998) have all reported to be in part genotype dependent. The fact

that on the same medium some genotypes produced embryogenic calli and

regenerated plants while others were completely incapable of regeneration

suggests that there are genetic components controlling this trait. Several

researchers have attempted to find location among the wheat genome responsible

for favourable tissue culture responses.

Chromosomes 7B, 7D and ID (Galiba et al., 1986), IRS (Langridge. et al.,

1991). IBS. 2BS, 6BL (Felsenburg et al., 1987). 4BL (Mathias and Fukui. 1986,

Mathias et al.. 1988), 2 DL, 2 AL, 2BS and 2 BL (Kaleikau et al., 1989a,b; Ben

Amer et al., 1992), mitochondrial DNA (Rode et al., 1986) or even cytoplasm

(Mathias and Fukui, 1986) have all been identified as possible regions responsible

for better callus induction and regeneration potential. Some others reported

different genes like reduced height/gibberellic acid insensitivity genes, Rht/Cai

(Mathias and Atkinson, 1987) semi-dwarfism allele, Rht 8 and day length sensitive

allele ppd 1 (Ben Amer et al, 1992) etc. to be responsible for in vitro responses.

It can be suggested that one or more of these genes are being expressed in the

highly regenerable genotypes on, one of the media (Fennel et al, 1996). Lazer

1981 also found genotype-medium interaction to be responsible for better in vitro

response.

Present course of study also supported the view of others that callus

induction and regeneration both was found to be genetically affected. In contrary

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Discussion 69

Vasil (1987) and Redway et al. (1990) hypothesize that differences in cultivar

responses are physiological in nature and can be overcome by culture conditions

and media. However trend of responding of media composition for callus

induction, regeneration, number of shoots per calli or root formation were in

general found to be uniform, irrespective of the genotype.

5.7. WIDE CROSSING AND EMBRYO RESCUE

During the last four decades, there has been an extensive use of wild

relatives in wheat breeding programm, particularly outside the country. Recently,

the breeders are using these as the valuable source of variation for wider

adaptation, tolerance to biotic and abiotic stresses and finally for yield

contributing traits. However, this is not an easy job, since many factors are

involved in between to obtain the hybrids. There are even some genes like Krl,

Kr2, Kr3 and Kr4 located on chromosomes 5B, 5A, 5D and lA respectively,

which are known to reduce crossability with many species (Koba and Shimada,

1993).

In present study, greater emphasis has been paid to callus mediated embryo

rescue, as direct germination of the rescued embryos was reported in the literature

to lead precocious germination resulting into small, weak, lean and thin plantlets

(Ozias-Akins and Vasil, 1982). Therefore, the crosses which are difficult to make

and where the seed setting is very poor, even one successfully cultured embryo

may give rise to several hybrid plants and the chances of getting an amphiploid are

therefore much more (Immonen, 1996). In addition to alien genetic transfers

mediated by callus culture promote multiple cytological variations and could also

be exploited as an adjunct to Ph- mediated Fj introgeneric hybrid genetic

exchanges (Mujeeb-Kazi and Asiedu, 1990).

Ter-Kuile et al. (1988) supported the view by regenerating Fi hybrids of T.

aestivum x Ae. variabilis and T. turgidum x Ae. variabilis and demonstrated seed

set on an otherwise anticipated self sterile population. Moreover, the Callus

induced doubling may prove advantageous for combinations otherwise hard to

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Discussion 70

double and also reported induced alien exchanges in wheat x rye hybrids (Lapitan

etal, 1984, 1986).

The hand pollinated spikes were sprayed with 2, 4-D + GA3 solution which

had a positive influence on developing seeds and embryo formation. In several

crosses with wheat x H. bulhosum and wheat x maize, Inagaki (1986, 1998)

could enhance embryo formation and then seed setting by spraying 2, 4-D +GA3

just after pollination. . This could be the reason of having better dissectible

embryos than in the experiments laid down earlier (unpublished).

In most of the crosses performed in the present investigations, the embryos

were rescued at 10, 12, 14 and 16 days post pollinations. 10 and 12 days old

embryos gave better callus induction than 10 or 16 days embryos. The possible

reasons for such behaviour may be that 10 days old embryos may require an

additional nutrition (Raghavan, 1976) or physical injury due to very small size

during dissection. On the other hand, most of the older embryos (> 16 DPP)

usually got distorted (may be due to initiation of degeneration) and callus

induction from such types of embryos was reported in very few cases. During the

present study, we did not need to enrich the medium instead 12 or 14 days

embryos could be cultured successfully to get regenerants.

Most of the calli regenerated on media containing kinetin (2 mg/1) or BAP

(2mg/l) + lAA (0.5mg/l) that led to produce multiple plantlets in many of the

crosses.

The calli in some of the hybrids like Bijaga Yellow x T. boeticum crosses

oi Ae. squarrosa (male) with Raj 3765 and Gulab, Ae. cylindrica (CD) with all the

five durums and T. araraticum (male) with Raj 1555, PDW 215 and PDW 233,

turned brown and no further development occurred in such cases. Also, in other

hybrids in which the wild species were taken as male parents namely, Ae.

speltoides (male) with HD 2329, Gulab and PDW 233, Ae. cylindrica (male) with

PBW 343, HD 2329 and Raj 3765 and all the hybrids involving Ae. triuncialis, the

calli became necrotic i.e. turned brown and did not respond at all to regeneration.

Seko and Nishimura (1996) and Fennell et al. (1996) reported this premature

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Discussion 71

rooting or necrotic phenomenon of calli to be genotypic character. Since the

female parent of these crosses do not have such characters, it may be the trait of

the alien genotype or a cumulative effect of some other factors.

Embryos of hybrids involving Ae. bicornis, Ae. caudate and Ae. lorentii did

not respond at all. This may be due to complete incompetence of the genotypes

used. However, T. boeticum, Ae. speltoids, Ae. ventricosa and T. araraticum

showed partial incompetence with PBW 215, Raj 1555, PBW 343 and Bijaga

Yellow and UP 2338 respectively, as hybrid embryos of these species were

reported to induce callus with other cultivars. According to Endo (1988, 1979),

the presence of gametocidal chromosomes in Aegilops species, causes sterility and

abnormality in certain genetic backgrounds. The Occurrence of abnormal embryos

was found to be a common phenomenon particularly in older embryos (> 16 DPP).

The hybrid plantlets established in the field were seen to exhibit

intermediate morphology, casually giving plants a profuse tillering. Having

intermediate type of plant morphology is important as it proves the validity of the

cross (Tyaukgva, 2000).

During present experimentation, embryo formation with hybrids involving

bread wheat varieties PBW 343 and WH 542 exhibited maximum excisable

embryos with most of the alien genotypes. Verma et al. (1999) also found PBW

343 and WH 542 to respond better than others in wheat x maize crosses which

may be due to the presence of 'Veery' blood in these genotypes.

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SUMMAHrAUV CONCLUSION

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Summary and Conclusion 72

6. Summary and Conclusion

Wild relatives of wheat are a valuable reservoir of genes which are either

absent in cultivated wheat or their diversity is narrow particularly with respect to

pest resistance, tolerance to biotic and abiotic stresses and various morphological

traits including the end use properties for making bread and pasta as well as other

products. Therefore, the wild species can be used as a potential source to add

novel variability and affecting the genetic enrichment program through wide

crossing. However, in most of the wide crosses the embryo aborts at various

levels of development. Hence, the technique of embryo rescue involving excision

and culture of embryos aseptically had been quite successful in transferring the

useful traits from alien sources to the background of cultivated species.

In lieu of this, an effort was made to standardize a regeneration protocol,

using the cultivars of both T. durum and T. aestivum species of wheat. Later on,

the embryos of the wide crosses were rescued to test the protocol on selected

varieties and media.

The salient results/observations recorded from the present study have

been summarized here under :

> Two varieties each for bread wheat (WH 542 and UP 2338) and durum

wheat (Bijaga Yellow and Gulab) were screened on four callus induction

media CI (MS+2,4-D, 2mg/l), C2 (MS + 2,4-D, 4mg/I), C3 (MS+ 2,4-D,

2mg/l + lAA, 0.5 mg/1) and C4 (B5 + 2,4-D, 2mg/l). The embryos were

excised at 10, 12, 14, 16 and 18 days post pollination/days post anthesis.

12 and 14 days calli of bread wheat and 12, 14 and 16 days old calli of

durum wheat were found optimum. Maximum frequency of callus

induction was recorded on C1 (embryogenic) than on C2 (loose and non-

embryogenic)

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Summary and Conclusion 73

> Five media viz., Rl (left on the same medium and exposing to light). R2

(MS + Kinetin. 2 mg/1), R3 (MS + Kinetin, 2mg/l + lAA, 0.5 mg/1), R4

(MS + BAP, 2 mg/1) and R5 (MS +BAP, 2ml/l + lAA, 2mg/l) were tested

to achieve regeneration from calli produced on CI, C2, C3 and C4 media.

> Three media viz., Rl, R2 and R5 were found to give good regeneration in

all the varieties, while R3 and R4 proved quite poor in this regard.

> The regenerated plantlets were grown on three media viz., SI (left on the

same medium where they were induced), S2 (MS + GA3 0.5 mg/1 +30 g/1

sucrose) and S3 (GR free MS + 20 g/1 sucrose) up to 3 to 4 leaf stage.

Among these, the S3 medium was found better than other two media.

> The regenerated plantlets were transferred on a range of rooting media

and the medium containing MS salts without vitamins + NAA^mg/l +

sucrose 20g/l) was found better responding than rest.

> The dark condition during callusing and light during regeneration phase

proved quite effective. Interestingly, the alternate light and dark

treatment using 20 watts electric bulbs (16/8 hrs light/dark) could induce

regeneration in calli produced on MS + 2. 4-D (2mg/l) medium.

> A temperature of 25±2°C was found optimum, while the further rise in

temperature resulted in the browning of callus.

> The plantlets were hardened in small pots filled with agropeat and soilrite

in equal proportions and put into growth chamber at 15±1°C and 90 per

cent relative humidity. Established plants were transferred in bigger pots

containing soil, sand, FYM and agropeat in equal proportions.

> Following the above mentioned protocol, six additional varieties each of

bread wheat (CPAN 3004, KRL 1-4, PBW 343, PBW 373, Raj 3765 and

HD 2329) and durum wheat (HD 4530, HI 8381, Raj 1555, PDW 215,

PDW 233 and MACS 2846) were screened to check both the validity of

protocol and selection of better responding genotypes.

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Summary and Conclusion 74

> The age of immature embryo, genotype, media composition and culture

conditions all affected the regeneration potential of the explant.

> The above mentioned protocol was further extended to grow hybrid plants

from the crosses involving wide relatives.

> Five varieities each of bread wheat (WH 542, UP 2338, HD 2329, PBW

343 and Raj 3765) and durum wheat (Bijaga Yellow, Raj 1555, Gulab,

PDW 215 and PDW 233) were taken as female and 20 species of wild

triticum and Aegilops species [T. urartu (A"), T. boeticum (A**), Ae.

speltoides{S), Ae. longissima (S'). Ae. sharonensis{S^^), Ae. bicornis (S ).

Ae. squarrosa (D). Ae. caudata (C), Ae. markgrafii (C), Ae. umhelluluta

(U), Ae. cylindrica (CD), Ae. ventricosa (D"), Ae. kotschyi (US), Ae.

peregrina (US), Ae. triuncialis (UC), Ae. ovata (UM"), Ae. lorentii

(UM' ' ) , Ae. triaristata (UM'), Ae. geniculata{\]M°), T. araraticum{AG)]

were used as pollen parent.

> The hand emasculated spikes were pollinated twice with pollens collected

from the alien species. The pollinated spikes were sprayed with 2, 4-D +

GA3 solution for four consecutive days starting from the day of

emasculation.

> The hybrid embryos were scooped out at 10, 12, 14 and 16 days with

slight variation in some crosses and cultured aseptically following the

above mentioned protocol.

> The embryos rescued after 12 to 14 DPP were found to give optimum

regeneration in most of the crosses.

> The regenerated hybrid plants were established in the field with various

levels of seed settings.

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BtBUOGKAPHY

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Page 138: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

ANNEXUKBS

Page 139: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Annexure I

COMPOSITION (mg/1) OF DIFFERENT MEDIA USED

I n g r e d i e n t s

NH4NO3

KNO3

CaCl2. 2H2O

M g S 0 4 . 7H2O

(NH4)2S04

KH2PO4

NaH2P04 .H20

IKI

H3BO3

M n S 0 4 . 4 H 2 0

i M n S 0 4 . H 2 0

' Z n S 0 4 . 2H2O

Na2Mo04. 2H2O

CUSO4, 5H2O

C0CI2.6H2O

F e S 0 4 . 7 H 2 0

Na2EDTA

I n o s i t o l

N ico t in i c ac id

P v r i d o x i n e - H C l

T h i a m i n e - l i c e

Glyc ine

S u c r o s e

PH

MS

1650

1900

4 4 0

3 7 0

170

0 . 8 3

6.2

2 2 . 3

8.6

0 . 2 5

0 . 0 2 5

0 . 0 2 5

2 7 . 8

3 7 . 3

100

0 .5

0 .5

0 .1

2 .0

3 %

5.8

B 5

2 5 0 0

150

2 5 0

134

150

0 . 7 5

3 .0

10 .0

2 .0

0 . 2 5

0 . 0 2 5

0 . 0 2 5

2 7 . 8

3 7 . 3

100

1.0

1.0

10 .0

3 %

5 .5

Page 140: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Annexure II

STOCK SOLUTION PREPERTIONS FOR MS BASED MEDIUM

INORGANIC SALTS:

C a t a l o g u e

MS 1 (10 x)

MS 2 (10 x)

MS 3 (1000 X

MS 4 (lOOx)

MS 5 (100 x)

MS 6 lOOx)

VITAMINS

C a t a l o g u e

Vi (lOOx)

V2 (lOOOx)

V3 (lOOOx)

V4 (lOOOx)

Vs (lOOOx)

1_

I n g r e d i e n t s

C a C b 2H2O

N H 4 N 0 3

K N 0 3

KI

C0CI2. 6H2O

K H 2 P 0 4

Na2Mo04. 2H2O

H3BO3

MgS04

M n S 0 4

Z n S 0 4

CUSO4

Na2EDTA

FeS04

5 0 0 m l

2 .2g

8 .25

9 .5g

4 1 5 m g

1 2 . 5 m g

8.5g

1 2 . 5 m g

aiOmg

18 .5g

8 4 5 m g

4 3 0 m g

1.25mg.

1.865g

1.390g

I n g r e d i e n t s

I n o s i t o l

N ico t in i c ac id

P v r i d o x i n e - H C l

T h i a m i n e - H C l

Glyc ine

100ml

I g

5 0 m g

5 0 m g

lOmg

2 0 0 m g

1 0 0 0 m

4 .4g

16 .5g

19.Og

8 3 0 m g

2 5 m g

17.Og

2 5 m g

6 2 0 m g

37.Og

1.69g

8 6 0 m g

2 . 5 m g

3 . 7 3 g

2 . 7 8 g

2 0 0 m l

2 g

lOOmg

lOOmg

2 0 m g

4 0 0 m g

I S t o r e d

At

10°C

10°C

10°C

10°C

10°C

10°C

S t o r e d a t

<40"C

<40°C

<40°C

<40°C

<40°C

Page 141: core.ac.uk · India has been a major importer of wheat grain till 1960s. It was with the advent of semi dwarf wheat varieties like Lerma Rojo, Sonora 64 and Sharbati Sonora, Kalyansona,

Annexure III

WORKING DETAILS OF GROWTH REGULATIONS

H o r m o n e

2 ,4 -D

lAA

NAA

Kine t i c

BA(BAP)

CA3

S o l v e n t

E t o n /

IN .NaOH

E t o n /

IN .NaOH

IN.NaOH

l N , N a O H

l N , N a O H

E t o n

D i l u t a n t

T c W a t e r

Tc w a t e r

Tc w a t e r

Tc w a t e r

Tc w a t e r

Tc w a t e r

S t e r i l i z a t i o n

CA

C A / F

CA

C A / F

C A / F

C A / F

S t o c k

s o l u t i o n

1 m g / m l

1 m g / m l

1 m g / m l

1 m g / m l

1 m g / m l

1 m g / m l

Liquid

s t o r a g e

0 - 5 0 ° C

-0°C

0-5°C

-0°C

0 -5 .0 °C

0-5°C

CA = C o a u t o c l a v a b l e w i t h o t h e r m e d i a c o m p o n e n t s

C A / F = C o a u t o c l a v a b l e w i th o t h e r m e d i a c o m p o n e n t s ,

h o w e v e r s o m e l o s s of ac t iv i ty m a y o c c u r .