The Discovery of Haploid Production by Anther Culture

Author(s): Sipra Guha-Mukherjee

Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 35, No. 5 (Sep. - Oct., 1999), pp.
357-360
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4293267 .
Accessed: 22/09/2014 21:54

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp

.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.

Society for In Vitro Biology is collaborating with JSTOR to digitize, preserve and extend access to In Vitro
Cellular &Developmental Biology. Plant.

http://www.jstor.org

This content downloaded from 157.181.127.228 on Mon, 22 Sep 2014 21:54:29 PM

All use subject to JSTOR Terms and Conditions
In Vitro Cell. Dev. Biol.-Plant 35:357-360, September-October 1999
? 1999 Society for In Vitro Biology
1054-5476/99 $05.00 + 0.00
TURNING POINT ARTICLE
THE DISCOVERY OF HAPLOID PRODUCTION BY ANTHER CULTURE
SIPRAGUHA-MUKHERJEE1
School of Life Sciences, Jawaharlal Nehru University, New Delhi- 110 067, India
(accepted28 May1999;editorI. K. Vasil)
I am delighted and honoredto have the opportunityto present a
personal account of my work on the culture of excised anthers of
Datura innoxia,which opened a whole new field of researchleading
to the extensive use of the techniquefor the productionof haploids
and theirapplicationin plantbreeding.The invitationfromProfessor
Indra K. Vasil, President of the InternationalAssociationfor Plant
Tissue Culture& Biotechnology(IAPTC&B),to preparethis Turning
Pointarticleis gratifyingandparticularlywelcomebecauseProfessor
Vasil and I both had the privilegeof studyingunderthe guidanceof
the famed plant embryologists,ProfessorsP. Maheshwariand B. M.
Johri,at the Universityof Delhi.
This article, marginallyautobiographical,is primarilythe history
of the productionof the first haploids throughanther culture, and
how this workaffected my professionallife. The Departmentof Bot-
any at the Universityof Delhi had become a world-classcenter of
plant embryologyin a shorttime underthe leadershipof P. Mahesh-
wari. Much of the research in the departmentwas directed toward
classical embryologicalstudies. In his famousbook, "An Introduc-
tion to the Embryologyof Angiosperms,"P. Maheshwari(1950) had
devotedan entirechapterto the emergingdiscipline of experimental
embryology.In it he discussed the control of fertilization,embryo
culture,inducedparthenogenesisand haploidy,productionof adven-
tive embryos,and induced parthenocarpy.For me this was the most
interestingchapter,in which he narratedhow the inductionof hap-
loidy can greatly facilitate the task of a geneticist in producinga
homozygoustrue breedingtype which otherwiserequiresa long and
laboriousprocess of back-crossing.
Like me, a small group of graduatestudents had been greatly
influencedby the book and showed a keen interestfor researchin
experimentalembryology.This led to pioneeringresearchon the cul-
ture of floralorgans,such as anthers,pollen, ovary,ovules, embryos
and endosperm,intraovarianpollination,and in vitropollinationand
fertilization.My own doctoralresearch,underthe directionof B. M.
Johri, dealt with tissue culture of onion flowersto study the physi-
ology of fruitand seed development.
My firstpostdoctoralpositionwas in the same departmentwith S.
C. Maheshwari,to study the biochemistryof meiosis. For this pur-
pose, I had placed in culture the large anthersof Datura innoxia.
After 6 weeks, on a hot day in June 1964, I decided to discard my
cultures as they all appearedto be dead. I was greatly surprised,
therefore,to observesmall plantletsarisingfromthe anthersin some
of the cultures.I removedone such anthershowinga small plantlet
with a slightamountof callus anddissected it. The plantletappeared
tFax 91-11-616-5886; E-mail:sipra@jnuniv.ernet.in
357
This content downloaded from 157.181.127.228 on Mon, 22 Sep 2014 21:54:29 PM
All use subject to JSTOR Terms and Conditions
to arise frominside the now dead antherwall. No otherliving tissue
was visible. AlthoughI did not have any direct evidence at the time,
I was intriguedby the possibility that the plantlets may have arisen
frompollen grains.
At aboutthe same time, I was guiltyof accidentallyruininga swing
bucket rotorof the MSEcentrifugein the laboratory.I had no option
but to informP. Maheshwari,whowas then visitingthe UnitedStates.
In an attemptto lessen my punishment,I startedmy confessionwith
the news that I had obtainedplantlets from the culturedanthersof
Datura, which may have arisen from pollen grains. I was not sure
how that would affect my career/punishment.Within a fortnightI
received an excited note fromP. Maheshwarisaying that I must as-
certain the origin of the plantlets, with a hint that I would then be
pardonedfor myunintentionalcrime.Soon,ourfirstpaperdescribing
the regenerationof plantletsfromculturedanthers,withoutconfirm-
ing their origin frompollen grains, was published in Nature(Guha
and S. C. Maheshwari,1964).
I devotedthe next 2 years to tracethe originof the plantletswhich,
I was convinced, had arisen from pollen grains. I found almost all
stages of developmentof somatic embryosfrompollen grains, with
partiallyor fully intact exines. The clinching evidence came from
chromosomecounts fromroots that confirmedthe haploid natureof
the plantlets.Thus, the second reportpublishedin Nature(Guhaand
S. C. Maheshwari,1966) confirmedthe originof the haploidplantlets
frompollen grains of Datura. A moredetailed paperwas published
in Phytomorphology (Guhaand S. C. Maheshwari,1967).
Besides S. C. Maheshwariand myself, the person who was most
excited was P. Maheshwari.His dream had come true. One of his
earlierstudents,IndraK. Vasil, had workedon the cultureof excised
anthers of Allium cepa, Rhoeo discolorand Cajanuscajan, in order
to understandthe controlof meiosis and pollen development(Vasil
1957, 1959, 1963). Vasil had left for the United States in 1962, and
our research project was in a sense the continuationof the effort
startedby him to study meiosis in vitro.Neitherhe norwe had made
any deliberateattemptto induce haploidyin culturedanthers.Earlier
attemptsto obtain haploids from the male gametophyteof gymno-
sperms (Tulecke,1953) and angiosperms(Yamadaet al., 1963) had
resulted in the productionof callus, but no haploid plants (Vasil,
1980). Thus, like many other discoveries and inventions, ours too
was by chance and not design, and with a bit of good fortune.
Since the discovery of haploid plants of Datura stramoniumin
nature(Blakeslee et al., 1922), haploidshave attractedthe attention
of geneticists, plant breedersand embryologists.By 1960, haploids
had been reportedin 71 species of angiospermsbelonging to 39
genera and 14 families (Kimberand Riley, 1963). However,the re-
searchand utilizationof haploidswerelimitedby theirlowfrequency
(Hu and Yuan, 1986).
358 GUHA-MUKHERJEE

SIPRAGUHA-MUKHERJEE
Born July 13, 1938 in Calcutta,India

Haploids are useful because (i) homozygousdiploids can be ob-
tained in one generationvia chromosomedoubling,whereasseven to
eight generationsare requiredto achieve this throughconventional
breeding, (ii) mutationscan be easily detected in haploids because
of the expressionof recessive characters,and (iii) gameticgenotypes
are fully expressedat plantlevel, therebyprovidinga wide spectrum
for selection in breedingprograms.
Earlier,some success had been achieved in inducinghaploidyin
planta.Forexample,KiharaandKatayama(1932) hadobtainedthree
haploidsof Triticummonococcumby exposingspikes to X-raysat the
time of meiosis. Muntzing(1937) had obtained a haploid plant of
Secale cereale by exposing the spikes to 0.30 C, whereas Norden-
skiold (1939) had achievedthe same resultby exposingthemto high
temperatures(41-42' C). Othermethodsused included distanthy-
bridization,delayed pollination(Kihara,1940), and hormonetreat-
ments. However,none of these methodshad proveddependable.In
his above mentionedbook,P. Maheshwari(1950) wrotethat"whatis
really needed is an agent for producinghaploid plants which will
give positive results with approximatelysame consistencyas colchi-
cine does in producingpolyploidy."
Therewas muchexcitementand prideamongmy colleaguesat the
Universityof Delhi in our achievement.In the summerof 1966, P.
Maheshwariwas preparingfor travelabroadand plannedto present
our workto the embryologistsof the world.However,destinywilled
otherwise.He unexpectedlydied aftera shortillness a few daysprior
to his departure.Had he taken this trip and announcedour workin
his magnificentand inimitable style, my destiny would have also
changed. I left for the United States within a few monthsafter his
untimelydeath, beforethe publicationof the 1966 paperin Nature.
I cannot leave the account of this period of my life without ac-
knowledging my appreciation for P. Maheshwari. He was an unusual
person in many ways with extraordinary sensitivity and understand-
ing, coupled with great oratorical skills. The wisdom of his advice
benefitted even the average mind. For 16 years he trained a large
number of graduate students in the Department, who later made out-
standing contributions and have shown their capabilities all over the
world. A hard worker and voracious reader, he had the laboratory
skill, scientific temper and the ability and insight to identify prom-
ising research topics. He had an infectious enthusiasm for science.
His ideas on experimental embryology put forth half a century ago
are now being pursued at many distinguished laboratories of the
world. He was thus a person far ahead of his time.
In 1966 I joined Robert Bandurski's group at Michigan State Uni-
versity. The University was home to many distinguished plant phys-
iologists and biochemists, such as Norman Good, Anton Lang, Joe
Varner, Hans Kende, Philip Filner, and J. A. V. Zeevart. I talked to
many of them about my work on the experimental production of hap-
loids. However, in those early days of excitement about nucleic acids
and plant molecular biology, my work did not interest anybody and
I accepted my fate.
In 1968, after I returned to India, M. S. Swaminathan, the then
Director of the Indian Agricultural Research Institute, gave me en-
couragement and the opportunity to work on rice haploids. In the
same year, Niizeki and Oono (1968) had described their success in
the induction of rice haploids from anther culture. I followed their
protocol and realized that the Indica varieties of rice were very dif-
ferent from Japonica varieties in their ability to produce plantlets
from anthers. I tried anthers from several varieties of Indica rice and
found positive results only from three (Guha et al., 1970; Guha-
Mukherjee, 1973). These studies established that gametophytic dif-
ferences play a crucial role in influencing the productivity of plant-

This content downloaded from 157.181.127.228 on Mon, 22 Sep 2014 21:54:29 PM

All use subject to JSTOR Terms and Conditions
 TURNINGPOINTARTICLE
lets in antherculture.This observationreceivedwide acceptancein
comingyears.
Haploidsfromtobacco antherswere reportedby J. P. Nitsch and
C. Nitsch (1969), three years after the publicationof our work on
Daturahaploids.But, surprisingly,no mentionwas madeof ourcon-
tributionin their paper althougha referencewas made to the work
of Niizeki and Oono(1968), which did acknowledgeourwork.I had
greatrespectforJ. P. Nitsch, but was verymuchdisappointedby this
blatantomission. I wonderedwhetherthis was because the western
worlddoes notpay muchattentionto contributionsmadeby scientists
in the developingcountries,or whetherit was morea personalchoice
and decision.
In 1970, I joined the Departmentof Biosciences at WestVirginia
Universityin the United States. I presenteda seminaron the pro-
ductionof haploidsthroughantherculturebut failed to generateany
interestin this area, as it was not in vogue at thattime. However,my
dampenedspirits rose when I received an invitationin 1970 from
the greatGermangeneticist, GeorgeMelchers,Directorof the Max-
Planck Instituteof Plant Biology at Tubingen,to attend an EMBO
conferencein Italy.He providedme a platformto talk aboutmywork
for the first time in an internationalscientific forum.He, himself,
showedgreat enthusiasmand urged me to continue this line of re-
search (Melchers, 1974). By now, there was much discussion and
excitementaboutthe potentialof haploidsderivedfromculturedan-
thers, and a numberof laboratorieshad begun workingon different
plant species. An internationalsymposiumon "Haploidyin Higher
Plants-Advances and Potential" was held at the University of
Guelph,Canada,in 1974, to discuss these advances.N. Sunderland
fromthe JohnInnes Institute(UnitedKingdom),delivereda lecture
on antherculture as a means of haploid induction,in honorof our
work,and as a tributeto J. P. Nitsch, who had unfortunatelydied in
an ocean diving accident just priorto the conference(Sunderland,
1974).
All this happened in an era in which women scientists had to
strugglevery hard to keep active and be recognizedfortheir contri-
butions(evennow I have doubtsif that era is over).This was a global
phenomenon.The professionaltalents of womenscientists were ex-
ploited to promotethe work of men, but they themselves were not
given the opportunityto develop their own professionalcareers.The
ridiculousstatementcould alwaysbe heardthat the wife of a faculty
membercould at most be kept academicallyalive but not hired in
the same institutionin a positionthat she deserved.In mostpartsof
the world,women'sorganizationshave tried theirbest to abolishthis
greatinjustice, but have not been able to cut much ice in the male-
dominatedacademic world.Even Delhi University,which boasts of
producinga numberof brilliantwomenscientists, has had only one
womanfacultymemberin its BotanyDepartmentin the last 50 years!
Fortunately,there are exceptions to all rules. I am, therefore,
greatlyindebtedto M. S. Swaminathanfor recommendingme for an
invitedfacultyposition in the newly opened JawaharlalNehruUni-
versityat New Delhi, which was meantto be a NationalUniversity.
It was a greatchallengefor me to be one of the five facultymembers
to start the new School of Life Sciences. It was an opportunityto
consolidatemy trainingto start a modernline of researchon plant
sciences. Webegan with studies on antherculture(Jain, 1978), pro-
toplast culture (Bhalla-Sarin,1977) and regulationof enzymes of
higherplants (Yadav,1974; Sihag, 1977; Suri, 1978; Talwar,1979).
LaterI focusedmy attentionon twomajorproblems:(i)understanding
the inductionof differentiationin tissue culture at the biochemical
This content downloaded from 157.181.127.228 on Mon, 22 Sep 2014 21:54:29 PM
All use subject to JSTOR Terms and Conditions
359
and molecularlevel, and (ii) the developmentof useful transgenics
in legumes.
At the XV InternationalCongressof Genetics, which was held in
New Delhi in 1983, I met ProfessorHu Han, Directorof the Institute
of Genetics of Academia Sinica, China, who praised our work on
anthercultureand remarkedthatit was responsiblefora substantial
change in the agriculturaleconomy in China. This was one of the
greatestmomentsof my life. He invited me to attendan international
symposiumon genetic manipulationin cropswhichincidentallywas
the 3rd InternationalSymposiumon Haploidyheld at Beijing,in the
People'sRepublic of China, in 1984.
In China, I received a tumultuousreceptionfroma large section
of scientists involved in workon haploid induction throughanther
culture. Yet anothergreat momentin Chinawas when ProfessorTre-
vor Thorpe(Canada)asked me if I was thrilled to see how our work
has grown.Chinesescientists have done excellent workon induction
of haploidy in higher plants. They have obtained haploids for the
firsttime in manyplant species and improvedtechniquesto enhance
the frequencyof inductionin many cases. Further,they conducted
studies on the cytologyof androgenesisand genetic variationof prog-
eny of pollen plants. New varieties were producedvia haploids in
some importantcrops, including wheat and rice (Hu and Yuan,
1986).
From the middle of the 1980s, work on genetic engineering in
plants and transgenicresearchtookthe driver'sseat in applied plant
biology. Studentsjoining the Universityduring this time and later
mostly wanted to work in this field. Thoughtissue culture still re-
mained a powerfultechniquefor the improvementof crop plants, it
was pushed to the back seat in favor of transformationresearch.
Nevertheless,workon antherculture is still being pursuedin many
countries for its applied value. Morethan 2000 publicationshave
appearedon androgenichaploids, including many books. In India,
where tissue culture laboratoriesstartedemerginglike mushrooms,
anthercultureremainedone of the majorobjectives.
Ironically,antherculturefor crop improvementhas neverbeen on
the agenda of the Indian Government.Therefore,the advantageof
our discoveryof haploidinductionwas lost. However,science is uni-
versaland therefore,whereverand wheneverthis techniquehas been
applied to crop improvement,I have felt gratifiedand recompensed
with a greatsense of satisfaction.
ACKNOWLEDGMENTS
I amgrateful
toProfessorS. P.Bhatnagar,
DepartmentofBotany, University
of Delhi,forhis valuablesuggestions of this manu-
duringthe preparation
script.I thankmycolleagueDr.P.K.Yadavforgoingthroughthemanuscript.
I alsothankDr.SanjeevKalia,Dr.JayantiSen,andmystudentsMadhusudan
DeyandSagarGhoshfortheirconstanthelp.
REFERENCES
Bhalla-Sarin,N. Studieson isolationandfusionof protoplasts.
Ph.D.thesis,
Jawaharlal NehruUniversity, NewDelhi;1977.
Blakeslee,A. F.;Belling,J.;Franham, M.E.;Bergner,A. D.A haploidmutant
in theJimsonweed,Daturastramonium. Science(WashDC)55:646-
647; 1922.
Guha,S.; Maheshwari, S. C. In vitroproduction
of embryosfromanthersof
Datura.Nature(Lond)204:497;1964.
Guha,S.; Maheshwari, S. C. Celldivisionanddifferentiation
of embryosin
thepollengrainsofDaturain vitro.Nature(Lond)212:97-98;1966.
Guha,S.;Maheshwari, S. C. Developmentof embryoids
frompollengrainsof
Daturain vitro.Phytomorphology 17:97-98;1967.
360 GUHA-MUKHERJEE

Guha,S.; Iyer, R. D.; Gupta,N.; Swaminathan,M. S. Totipotencyof gametic
cells and the productionof haploids in rice. Curr.Sci. 39:174-176;
1970.
Guha-Mukherjee,S. Genotypicdifferences in the in vitro formationof em-
bryoidsfromrice pollen. J. Exp. Bot. 24:139-144; 1973.
Hu, H.; Yuan, H., ed. Haploids of higher plants in vitro. Beijing: China
Academic Publishers;Berlin:Springer-Verlag; 1986.
Jain, S. M. In vitro productionof haploids in higher plants. Ph.D. thesis,
JawaharlalNehruUniversity,New Delhi; 1978.
Kihara,H. Formationof haploidsby meansof delayedpollinationin Triticum
monococcum.Bot. Mag.Tokyo54:178-185; 1940.
Kihara,H.; Katayama,Y. On the progenyof haploidplants of Triticummon-
ococcum.Kwagau2:408-410; 1932.
Kimber,G.; Riley, R. Haploidangiosperms.Bot. Rev. 29:480-531; 1963.
Maheshwari,P. An introductionto the embryologyof angiosperms.New York/
Toronto/London: McGraw-HillBook Co.; 1950.
Melchers,G. Summation:Haploid research in higher plants. In: Kasha, K.
J., ed. Haploidsin higherplants:advancesand potential.Proc. First
Intl. Symp., Universityof Guelph, Guelph;1974:393-401.
Muntzing,A. Note on haploidrye plant. Hereditas23:401-404; 1937.
Niizeki, H.; Oono, K. Inductionof haploid rice plant from anther culture.
Proc. Jpn. Acad. 44:554-557; 1968.
Nitsch, J. P.; Nitsch, C. Haploid plants from pollen grains. Science (Wash
DC) 163:85-87; 1969.
Nordenskiold,H. Studiesof a haploidryeplant.Hereditas25:204-210; 1939.
Sihag, R. K. Regulationof nitratereductase level in Pisum sativum.Ph.D.
thesis, JawaharlalNehruUniversity,New Delhi; 1977.
Sunderland,N. Anthercultureas a meansof inductionof haploidy.In:Kasha,
K. J., ed. Haploids in higher plants: advances and potential.Proc.
First Intl. Symp., Universityof Guelph, Guelph;1974:91-122.
Suri, S. Studies on stress in relationto nitrate reductase.Ph.D. thesis, Ja-
waharlalNehru University,New Delhi; 1978.
Talwar,S. Studies on gibberellininduced enzyme release in barley seeds.
Ph.D. thesis, JawaharlalNehru University,New Delhi; 1979.
Tulecke, W. R. A tissue derived from pollen of Gingko biloba. Science
117:599-600; 1953.
Vasil,I. K. Effectof kinetinand gibberellicacid on excised anthersofAllium
cepa. Science (WashDC) 126:1294-1295; 1957.
Vasil, I. K. Nucleic acids and survivalof excised anthersin vitro. Science
(WashDC) 129:1487-1488; 1959.
Vasil, I. K. Some new experimentswith excised anthers.In: Maheshwari,P.;
Rangaswamy,N. S., ed. Planttissue andorganculture-a symposium.
Departmentof Botany,Universityof Delhi, Delhi; 1963:230-231.
Vasil,I. K. Androgenichaploids.Int. Rev. Cytol.Suppl. 11A:195-223; 1980.
Yadav,N. S. Photoreductionof nitrate reductase;its mechanism.M. Phil
thesis, JawaharlalNehruUniversity,New Delhi; 1974.
Yamada,T.;Shoji,T.;Sinoto,Y. Formationof calli andfree cells in the tissue
cultureof Tradescantiareflexa.Bot. Mag.Tokyo76:332-339; 1963.

This content downloaded from 157.181.127.228 on Mon, 22 Sep 2014 21:54:29 PM

All use subject to JSTOR Terms and Conditions