Artificial germination of Sorghum vulgare Pers. Pollen

Item Type text; Thesis-Reproduction (electronic)

Authors Humphrey, David Ford, 1934-

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Download date 25/09/2021 11:53:46

Link to Item http://hdl.handle.net/10150/551560 MLTIF.IGIkL GERBOZATIGE . . . op v; . '■ ■■

SOHGHUM WLGAEE BES» POHEN

fey ' '

. David Ford Hwaphrey

& ffeesis Submitted t®. the Fasti.ty ©f the

. DEPARTMENT 0F PEAIMT BREEDING

In Partial Fulfillment ®f the. Eequirements , _ ... For the Degree of ;

MSTER OF'SCIENCE

In the Graduate College

.SHE UNIVERSITY CF-ARIZCEA STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at The University of Arizona and is deposited in The University Library to be made available to borrowers under rules of the Library*

Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made* Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in their judgment the proposed use of the material is in the interests of scholarship* In all other instances, however, permission must be obtained from the author*

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

o -j?

Professor of Plant Breeding > e '

ii ; ■ : . • : AGKNCELSDGHENTS

The author wishes io eapress his sincere appreeiaSieB to

Dre Lee Se Stith ;©f the- Department of Plant Breeding for eon# tributing helpfnl adviee^ suggestionsg and assistance during the preparation of this manmseripte

Appreeiation is also extended to the Botanj Department for mse of laboratory faeilitles and equipments To Dra Bebert

Harris of the Botany Department my heartfelt thanks for the photomierographs presented in this paper and also for the many rewarding diseussioms® ' ' ' - '

To my eQlleagne2 Mr0 John Nelson^ sineere gratitude is extended to him. for his aid in the preparation of portions of this thesis*. -

To all my fr±@ad% who in any w y assisted me in the ' writing of this manuscripts thankse

ill ■r. TABIE OF CONTE1HS

Page

fflTRODUGTlOlo O O O O O O O = O O O O o o o O O 1 LITERATURE REVIEW » =• O O & O O © © ■ © o o o o O O • o O © , - '■ ^ , ;,-4- ' : m m i i m methods © b © ,p' © ©‘ o o o' o o o ° ° 7 Sourceof Pollen ©■ o o © © © o o © o. o o "is : Test' "OondltloBS o . ©. O © p © ©» O O O O O o IS , SialnabijLity 0 o Po o O Os o © o- O o,.o 0 o © o o o o 15 Attempts at Germih'atiori"' and 'Seseription' of Methods o © 16

Sitheits: aid, FoIIination: o . © , O . O'© d © 16

Germination Under Natural'Field Conditions ■ o

Germination Under.Greenhouse Conditions = » o ; i l . " - , Chemical' Solutions nsed on Eollen" Grains » « » i 18 .FlSH.'b SXbrBGtiS" © o © © /© © © o o ; © o' © o o o © o 19 Ghemieals xn Agar.o » o- *, o * * « * * o,- . o o: o e O © ; : 21,

Paraffin Stmdibg' o 0"o 7 o'''o: F' o'"o'. v ; ;'7V © o o o 21.

Effeet of Mght bn Pblleh Germination in vivo © © 22 Effect of Light en Pollen Geminatibn" in t o O l e l d o * ' 25 . / The'Pollen. Grains'Expbsed’to Air « o Z l 6 o * 7 * © © 2# tr»T~jpvt jtii *y"T*r cyt J** * • *' -■ — ' v - j j ^ - y ' . ' » - i* - * ' ■* •' u j%E*DUlaU|* © © © O . ©A O © ;© O © © d'. > O' © p . © © > © O 0 0 , 0 © O d " 0 26 a l* - -I’ -» ^ * * • r ‘ » i- . StalhBiliif telt 7'7 7 © ©. o © o o ■ ©1 o 0 © 26

Studies nith Solutions © o © © 27 •PblIen'Gerainatx©n'7: '77 ' i 7 7 ‘ 7 I I I 1 I I J O © ■37; Hhmidity CSaiber'Results 7 7 ; » e 7 : 7' ©' ' o o o So : Light Control GHamSert o 7 ‘o q o. o o o. o © © • O' © 50 • Light Gontroiled'Field Study : 8 o : o o ,o 7 o © - d 51 Pollen Exposed to 'Air7 0 7 7. 0 = o 7 7 o.7 o A ss :BlBGtBSICEa.;'e » f- o o'; i..: 7 e 7 : 7: 7;7. 7 7 A./ i S6 .m g 'goncsjsiono o © 55 0 o © o O o © o © 55 Observations b ■' ; © ' d . 6o ;■ © o - o o © o b. o o o o 7 © 56 MIBmiUEB CITED © O O . o o 58

'7

iv LIST OF TABLES

Table Pag®

1 OonceHtrations ©£ Sugar and Salts Salt and Gelatin^ and. Plant _Exfcraet0 . Tueson9 .Arizona « •July5 i^6l» «• 0 O C 0 *:». O, 0 0 0 0 0-0 0 © ffi 20

2 Per Oent Humidity at Given’Temperature Within a Glosed Sp.aee When an Exoess . of the Substanee ' Indicated, is in Gontaet with a Saturated Aquecus Solution of the Gifen Solid Phase » © s . ©. © o © 23

3 Per Gent of Stainable . Pollen Grains for 8 day Periodo Tueaons Arizona > August$ 1961 © . • ©' = o 29

k Per Gent; Belative Humidity .in Sorghum Field at Campbell Avenue Farm© ■ Tucson5 Arizona „« .August* 196l e 0 0 O O O 0,'. 0 O 0,0 0 0 0 © O O . 0 O O O 0 < 30

5 . Temperature (0pe.) in Sorghum Field at Campbell . Avenue'Farme -; Tucson* Arizona -,*». August^; l96io- © © 30

6 Comparison of Temperature (0G

T • Comparison of Seed Weight of - Sorghum Seed Pro# duced Wnder Efermal Pollination Bags and Black ; iagse _ Tucson* Arizona » September* I96I0 .0, 0; 0.'A A3

':fp- LIST OF FIGURES

Hgu^e - . Page

1 Shewing relationship (average) between stainable goilems relative :hui8ieiitys an4 temperature for . eight' dayso Tuesons Arizona « July^, 1961=, o l » 31

2 Per sent stainable pollen grains for each of five ■■■■’. dajsc, Tuesons Arizona •» Julys 1961a 1 ». => o -o « => 32

3 Showing daily relationship between stainable pollen grains„ relative humidity9 and temperature under field eonditions o Tue sons ^riz ona e <, =, o «, e 0 0 o 33

4 Showing daily relationship between, stainable pollen grainss relative humiditys and. temperature under field GOBditibnSd fues©ns Arizona 0 o » ® 0 « @ 0 ®, 33

5 Showing daily relationship between stainable pollen grains g relative humidity^ ‘and temperature under field conditionso luesons Arizona 2 > 0 o » » » ® o 3H

6 . Showing' daily relationship between stainable pollen 1 grainsg relative humidity^ and temperature under field conditions^ Tucson. Arizona . «, „ e 0 0 ® o ® ; 3k

7 Showing daily relationship between stainable pollen • grainsg relative humidify =, and temperature under . • field ooaditionse Tucson. Arizona o o e e o * '» Q e 35 ■ . :: ' ' " \\\ ; ' ' 8 , Showing daily relationship between stainable - poUen grains^ relative humiditys and temperature under . field conditionso Tucson^ Arizona » „ 0 =, . . = <, » 35

9 Showing daily relationship between stainable pollen grains^ relative humidity^ and temperature under field condition So ' Tucson g Arizona » <,'«■■«; 0 0 e «, 0 36

10 Showing daily relationship between stainable pollen grainsa relative humidityg and temperature under field condition So Tucson. Arizona » 0 e •» . o'* 36

11 Eelationship of per cent pollen germination to per cent of total pollen grains that shrink upon ex* pesmre to aire Tucsons Arizona ». Augustg 1961 « «, 0 kS

. ‘ ' v i : ' ■; iism op .

Elate Page

1 (Soggfcim Tolg^e) slowing(1) '(2 ) eMne'^'' fakre pere^ and (Ui the. start of'pollm tube' . growtho , X; 1000 o- O' © 0 O O O O O 0 © © -■ . ©--- -V-V © -y © 6 38

2 - (Sorgiatam TOlgaye) pdHen iuBe/ ggewlfog' perieliiiial ' '' t© tiie exme o X. 800 o o a © • ©■ © o © © - © - © © © © © 38

: 3'■■ terminating pollen tubeQ. X 600© © © © © © © © . © © 39 ? - ■- r - - , - v ... . , . , ■ - ' . . ■: : ■ ■ : ' . : -■ ■: ; k Mature pollen tube© X 500 © © © i © © © © © © © © 39

, ■' v i i BMEWSIO!

urssis sergaroBj, aerem*..va&gar@ :\&gasr®ois o^ajag aua«3ug@®@m@ to E@p^ m s ®a@Bg the earliest ®f il M plmis is Ibe dLemesidLeatei. aad latilised as hmaam feed aad as feed for iiwstoek (Martia ■2,939}®

Qraia ser^i* is aa aaattala m m s@as©as shert^dgg-g. aitesgea® pesitiw grass plant grew on all @®®tia«ts ea a total of mere than

88 Billies seres b jfiihemgh grsw as aa aanmali, s e r ^ w tea bmu aaiataimed is a germaial f@r as leag as six years la - the field im

Seathezm Calif©rai® and for thirteem years in a . gr@emhe%@® (?iaal> 1

Siephmsp. . and Martin 1936) and (Martim Ifljl m d 19$l) o ScMP^tom is grom #iefly f@r grain tet the stalks are lmg)ertmt . as £@rag@o fh@

erashed from the stalks @f @met serghm varieties is need t©

, f® ftilf appreeiat® sad mderstaad s®rghwa9...„th@ reader shenlS, have a tetanieal h#@kgr©nnd @f # e planto S @ r ^ m : hel@ngs t# the family .Sraaaiiieae$> tribe jtodrepegenaeo The eal^yated, sorghms b®l@ng t@ @a#_ ^pe@iess Ser#mm ..'rolgare^ %Ai@h iB^mdes,. sneh diverse types mi the f@[email protected] (1) s o r ^ w f@r .grain (grain s©rghm)p (S) s®rghw fer fiber (brew. @@rm)g (3) serghw f@r grass (mdaagrass)p and (k) serghwt f©r ferag# (@©rg©)o She grass serghms are too kindsj the mltivated #mm@l# smdaneae©) ©essmonly kn@m as md@mgr&@@p and the perepaial halepense) ©@»©aly te@m as Jehnsengrass

(jftrtashenger 29k®) am# (tfard 3961)6 $1® sergtam plaat. is a ©ears® amm@3L ©mlms tee t® fifteea feet md" semetises »re im Wighto The swfaee ®f the ©timss sheaths^ aad leaws ar® glateettSo A leaf asls at e@@h Bede alternates ®n .©ppesite sides if the st@Be • The leave# appear in a plane© A ted Is

f@m@d at ®a@h md@ ®f the ©tijas esasept the terMnisl ©as® The 1b= ' fl@res©@n@e ©f ser^mm is a paaiele ©f varying sia® and density t-dth many teanehes tern ®a a hairy assis© The; sessile spikelets are p@rfe@t and mssally fertile tet the pedieellate florets are sterile @r stamiaat®© The le@fiaa and paisa are thin and. tr@B#la@emt and "fee lemma may ie either amei @r' amless- (¥iaals St^hea@s and Martin 19363©

ah@at eqmal diameter© The rests penetrate t® a writing depth ©f three t@ femr feet and gmerally a aariroa @f sis feet© The mnateay and ; swBphelegy ©f the serghm. plant is indeed a mest eenpleac shbj®©t| the purpose ©f the foregoing brief deseription is t© give a general pistnre @f the plant and its root system© Grain, sergtaa es&ibits a high degree ®f irbmght t©leran©@ during seedling establishment, and as a .mature plant© This @har.a@t@r<=> istie makes it especially uell- adapted to arid and seml^arid lands© Other desirable tearaeteristies possessed by grain sorghmy im addition te dreaght t@l@raa©es are its fibrems root system and sh®rt growing season Widh make the speeies valmable for reclaimed or re® el&lmlag desert land where water is a limiting' fa© tor © - Taribas methods ©f plmt improvement have evolved and been :SS@d in the United States the past, sixty years© Application of a particular breeding method is largely dependent mp®a the . genetieal behavior of the species G. Plant breeders have long been

interested in hybridizing varieties^ specie85, and even genera to

produce wide genetic diversitje Barriers to hybridization often

encountered. in breeding work include flowering of the selected parents

at different; timess failure of pollen-'grains to germinate on the '

stigmas failure ;©f the tubes; to grow through the style; .and bursting-1

of pollen grains on. stigma and in the style» A plant breeder should

become familiar with the growth^ morphology^ and reproduction prow

. cesses.; so that the' -breeding program can progress with ma2n.to.um

efficiency through timely application ©f proper techniques^

The objectives ©f this study are te artificially germinate

Sorghum vulgar© pollen and to describe environmental conditions that

are,favorable, or detriment'Wl' to. pollen -germination and. subsequent

pollen tube growth# Once the basic environmental factors have been

established^ the f easibility of storage and shipment of sorghum

pollen, grains can be determined# Physiological -studies can be con#

ducted and possibly some ofthe previously mentioned barriers to . V

hybridization ©an be esplaineds. ,, -

Possibly the storage and shipment of sorghum pollen could

result in a definite economic ■advantage® .. Where planting - space ' is a ;.

limiting factor in a breeding -or genetic program^ storage and shipment

" of pollen could facilitate and speed up studies in plant breeding and

genetic studies- throughout the world-p

// Positive 'results on the germination of pollens the effect -

of environmental conditions on pollen germinations and pollen tube

growth has not been reported for S e r # # vulgage0 The need for this t e w been maS®. .:t© establish en.vir©nmeBtal eoBdliioas Beeessary fm madimmii p®ll@a gemiaati@m a M pell@B '.i*e sb attempt las teeea. made t© devise a technique t© germinate sorghum pellea that

, eoBJLe ©@ ms©e pra@m©aju^ ma e r xxejbe ©©zi@xm©BSe> ■

fb@ very natyre ©f this stte#' t©' dewlep a satisfactory ■ teeteiqiae t@ germinat®. p©12.@B grata®: is tedla that the resalts miii r a m m E B wnsat-

W®a@k®ms@ (IfSf) Reports that la lS2ka iadel, @a Italiaa

astrommes aai. math@matl@l@aa shll® ©xaaltiiag Sii® pistillate

stigma of Partmlaee ®l®ggae®a netieed that ®a@ @f the hairs iemmated ia a poUea grain 0 M&.&1 is r@©@gaisei as the first

selemtist t© see a pellm grata ppedaee a tabe whieh eater@d the stlgmati© tissae aad gradually iis^-peared iai® # e style© Ariel reported that the pollea tetees ©leagate hit, hy bit mad fiaally

@©a@ M eoBtaet with the ®wle» fh@ earliest ebsesvatleas m polled tabe growth appears to b® those ©f Worn Mohla as @it@d by

?iss@r (195l)o Sehleigea and ®iegham9 as reported by Wfdeh©ms@

(1959)a stadied the to vitro geranmation @f pellem grains of several plants and ©oaelmded that the germination varied from aperies to aperies or even from variety t® varietyo, Mangin$ as iisomssed by W@deh©use (1959) 9. distinguished t # layers to the

; . ' , . pollen wallj the external etatiaiaed. laye%>he #^,ed the oadmo, and the internal layer which was f ©and t® consist of ©ellmlese en its eater sid@s he' called the tottoe^ ##c@rding to Wodtiwase (1959),

Biemrg® confirmed these ©bservatif^p and noted that early germination consisted ©f am evagenation of the intine©

A.©e®rdtog t® several investigatorss Goteh (1931), Erdtoaa

(19&.3 and 1952)s and Wodehons® (1959),'the pollen grains of the

Sramtoeae family are remarkably nniferm- to "the' morphological appearance ■ between- varieties or species 0 'Sorghna vtalgare pollen

5 , - :.... fedjy nateore is geaerally spherlealj in size it. vmges' from about.

21 miereBS . t© a litile ever 1@® mi#®ms 1m diameter^ ' ,

, . Weiehemse (1959) describes morpholegleal physiological characteristics coBceriaiag pollem grains. ©f the Sramineae family $

' • / -*$he ezine is t him am# lacks. imtirely ■ ; ' scelptpriag adornments of any kinds except a ' v V slight- hmt characteristically granmlar. t^tnreg- - -and; tiieir imiine is thiek m d hyaline» the l . ' interior ©f the grain is- generally tightly packed. ' : iwith small starch grains and possesses a. small;.. - hyalin®'which is diametrically opposite the perei It is to the expansion ®i‘ these materials ' and the thiek intin® "upon the' absorption of water , • . ' that the grains ewe their r owdish' contow when : ' ' ' 'moist©'5-

According, to Irdtman (I9 k 3 )s the grains may generally he termed: - ' ...... ; - - : . - v ■: v ; ■ . . '

:*Ib some speciess however^ the surface of the graia^is slightly, rough otring to local separation; of. the ektexine from the smooth, surface of th©- endexin© o If examined under a high revolving ;P@wer^ vthe: eatiiMs nearly : §lw^S;: se#> t© possess a ' ■ minmte' granular• ©r retieulate. teitirej at high' adjnsteientj, dark meshes surrounding' bright areas . at. lew'1 adjustment^ hri^t meshes. encircling darker - mat©riaio": ' ' " . : '- V' . -

The - ezimeg - likethat of 'the skim of ^animals9 is the- pr©-0 - . tection ®f the organism from injury hy external agencies such as excessiire desiccation^ - destruction hy lights and mechanical injury©

The ©sine protldes. for' • tw© .functions - which are the speeial pro» -

■perties of pollen • grains "by providing for the emergence of the pollm tube" at th® . time ®f poilinatioa^ and accommodating changes in volume as the grain takes • up -and . gives; ©ff moisture© . The erm© readily does this in response to the ever ©hanging.humidity of the macro m d micro climate (Srdtman 1952.)o ■ . . ; The artifieial gemlaaiiea sf ^©11@b ; fd.ls■ iai© three geaeral methods based ©n the nat&re @£ pollen .gralass (1) pollem f©r whieh water alone is neeegsary for g@rBiiBationa- (2) pollen that requires water and a ehemleal; st&mlamt hther than smgars and (3) pollen which;

require#' furiems . 8#3mti@ms smgar for germimatiom. (Amthomgr and .

Harlan 1920) and (Addieett 19h3)o Honaally pollen grains do not

germinate satisfaetorily in waterg however> •aqaeous solutions of smeros© with or without - addition of accessory'Jsabstanees produce good

results© Pollen of some plants may geminate easily under a wide

range of .conditionsj, while m others the requirement may "be very -

specifie© ' - ;

SnthoBy; and Harlan - (192@) were among the first workers to

artificially geminate pollen - in the • Srmineae family® Harlan noticed tiny drops of water on the stigmaM,© tissue prior to pollination in ..

the morning® This observation led to an experiment hereby barley pollen -sas germinated.® Pollen was.taken -from, an anther-ready to .-

dehisce and dusted on a slide inside a Van Tieghem cell© A piece of mesephyll from the leaf ©f the garden pea was placed in the cell to

supply Moisture®. inthony and Harlam reported'that they secured -

germination within five 'minutes on'•their first attempt©- -Earns (1915)

gives a complete.'outline on the correct procedure. on hew to use the

Van Tieghem cell for germination pu^oses© He also. found that if he

used a small 'amount @r gelatin in his artificial Aediumg germination

of pollen grains increased© Possibly gelatin supplies proteins® -

Firbash (1911) germinated. rye and wheat pollen on Stigmati© surfaces©

and on artificial medima© He-'concludedthat the humidity of the air j, a tempegatweg amd age e£ pollen affeets tfe® mability ©£ p@Uen» 3k the aytiflelal gemination of @ © m pollen^, Knoulton (1922) ©©ndroted a series of repeated ezperimemts ©n ©of per ©esat agar and 1$ per @emt sugar at a teaperatwe of 23e C© and 90 per ©eat relative humidity ©

Tenkatrsmn (1922) reported germination of sugar ©an® pollen was ob= iaiaed ©n artificial medium of Si 7 per ©eat agar and 26 per ©eat saereseo Bair sad loomis (19k!) also reported that they achieved germination of sugar ©an® .pollen @a 1©5 per ©eat agar and 25 per ©ent

886*086® •

iyyangar and Ea® (1936) studied, pollen germination and pollen tab© growth of Sorghm margaritlferam pollen ©n artificial medium©

Sowing pollen ©a kl per ©ent saerose agar3 they found that germination usually started within IS to 3© minutes and growth was completed in three or four heurso The rate of growth was not uaiformo. They also reported that -generally there was a preliminary period of growth at

a moderate r at®d one ef rapid elongation^ and a slowing downs the decline in growth feeing more gradual .than the early acceleration®

Results ©f pollen germination test’s on Sorghum vulgar® made at State

:Gelleg@5 Hew Heiieo during - the summer of 19kf fey Artsehwager and

McGuire (19&9) were reported as not @eadlmsiv@o They .observed

sor^nm pollen germination and pollen tub® growth in unopened anthers but because of crowding of the pollen grains9 the pollen tubes were

twisted and ©oUed® They also reported the nutrient medium r®=>

commended fey .iyyangar and Rao (1936) proved tansuitafel®^ .and ©on^-mded

that osmotic eonsentratioa of the medium made it unsatisfactory»

On© ©f the few articles w i t t m on the effect of time in :' , ' ■ ■ ■ : • : . - ■■ • ; , ; ?

regard to germination was written by EaghareB and Barash (1959) c In

this classical experiment,, pollen was used from areea palmse They

reported that pollen grew for a 2# hoar period® Three complete phases

- in the. germination .@f pollen grains and •growth, of : pollen tubes were .

•ebserredU - ,An initial lagphase lasting one to two hoars after sowing

the pollen on artificial medium was reported# - This lagphase was

' felloweS by a period of' stimulation generally extending over a period1

of/three to twelve hours# The final phase. lasted from twelve, to-

twenty-four hoursg and mo appreciable stimulation was recorded during

this period# In the final phase am increase in germination of only .

■ t w to four per cent was noted# Replication of this experiment gave

identical results e ■

According to Pope (1937) barley pollen will germinate, within

five minutes after falling oh a stigma tie hair and the gametes will

enter the pollen tube within ten minutese Stephens and Qninby (193%)

observed. that .sorghum pollen under, f ield conditions lost its viability

within five hours,, as seed set failed to materialize in heads dusted

with five hour old pollen# lyyangar and Rao (1936) using sorghum

pollenj, stored some packets of pollen in beakers in moist chambers

and some packets of pollen in open tubes' within a refrigerator at

1®® G# In the first experiment^ they observed about 28 per cent

germination after %8 hours# They reported that ..pollen Is capable of

fertilizing ‘up to the third days though, poorly®. According to Bair

and looms (19%!)^ under, optimum conditions for germination and pollen . \

tube growth of eorns the elongation of. the pollen tube Is sufficiently,

rapid to be plainly visible under the microscope e . '10

„ . Agsia^ aee«pdlag to;. Bate a ad £.o©miss ©a# major factor la the sueeess ©f g emanating poll® ©a a smgar agar medium is the imbedding of the poll@a grains o The agar p@r@eatagea t@!Kp@ratwes aad ©©©ling rate are Important !a this @esm@etl@@o Bair postulated that-appro^i^ matelgr tso-^teds of the grains should he iabeddedo .

. Towards the m d of the nineteenth ©mtury9 systematic study on the storage of pollen was oondaeted hy langins Eittin^i©msea and

Moliseh as reported hj tShdehous® (l^S^)o- ■ They investigated the longevity of pollen tf more than 80 species stored under air dry @©n» ditioffis. (fe^osed t® room twperatir®)® They investigated the offset of ®9 3©s ©08 and 90 per ©ent relativ® humidity m the viability @f the pollen at 0O These early workers ©eneluded that storage at lew humidities triples^ ©n the average9 the longevity ©f those pollens which it-affeetSo

According to various reports in the literature3 - Graaineae pollen till not retain its viaMlity unless the .relative humidity is between 7# and 9$ per ®®nto lati®ls as sited hy-Johri and Vasil

(1961) g stated that the pollen of g@a mays remains viable for ten days at ?@ 0O tith $0 to 7© per cent relative humidityo He reported negative results when the temperature was »5S Go tith less than $Q per ©ent relative .huBdiity©

Martin and ye e m (1915.0) ■ reported that germination of apple pollen was indueed most sueeessfelly on 2 1/2 ■ per cent sucrose agar ■ ■ . - medium from 22® te 25® Go T h ^ also reported that no secretion was found @n the .stigma at the time ®f pollimationa whereas^ Anthony and

Harlan (1920) femmd a water secretion on the stigmti© tissue ef "bsrley i© polliaat-ieno

fasil (1958) g@p®rt®d that at h&& hwiiities (@40 per

relative hiiiidits1) siered pell«a remins vialbl# £&s long periods o

Jt high Eroidities (60 per ©eat relative hmiidity and shove)s pollen

is often attacked hgr laeteria and fengi ^iaieh renders the samples

mfit for farther uses Bennett (1959) stated that Paspal#m dilatatGim

does set shotf any germination thirty mismtes after dehiscenee @f the

antherSo ■

Smith and 0©@hraa (1935) ©ondaeted e^eriments on the effect

of temperatmre m pollen germiaatioa. and pollen tmbe growth in

tematoeso- .They reported 66 per ©eat pollen germination after 6©

hours at a temperature ©f 85@ F0 At T©@ To germination m s almost •

as goedg hut at 5©@ Fo only fl05-per © m t gersdaatedo In some spe©ies9

pollen Bill retain its ability to geminate f@r some tim@o Apples

pearg and plum pollen will remain viable for several weeks or even

months (Fiery and Tomas 19&3)o

The viability of any pollen must be ©losely related to the

effect of. temperature and humidity© From the review of literature^,

the general conclusion is that the pollen ®f ©ramineae plants loses

viability soon after dehis@®n©@, ©f the anthers^ ©specially when

"temperature and humidity are unusually high©

In addition t@ studies related to relative humidity#

tetperatmre# chemical sabstan©@@# and age ©£ pollen grains# attention has been given to growth promoting mhstam©$s and natural plant ■.

prodmetse I&fferent morphological parts of the florets have been

used to stimulate germination and tube growth© Petals from apples and pears were placed im artlfieial medtom and pollea graiag iatee-

.dacedo//; SeiabisdldL (1##) m d % Jehrl sad'Tasil’

eeatiaied. that pollaa geraSmatien: siiMXa'ked by tM@ areaaii©' . '

... smbstaases #@WeiWd by the petals^ Mollsehg as sited fey J@3afi sad

Tasil1 • iad$©at@s pe3^m. graims themselTes are ■

Tided .aith. a ©©msiderafel®' amewfe ef food .material in the form ®f . .

earbehydraies and fats© ■ ■ '

' Smith (lfS9) stated "that three indole aeeti© acid im ooa^-. ■ ■ , '

©emtratiems ®f ©ne part per aillioa stimulated the germination of

pollen grainse The r ate 'of' elongation of the pollem tmfee was im»

' ' ' ...... ' .oreased as was the total :g.@rmiaatiom perei^itageo . Banliher (1938) -

also reported that m t h the addition of varying amounts of.;three

.indole aoeti© aoid^ m.o feeaefieial effect of germination m s ofeserwdo •

;. A. eonsiderable improvement im euXter© medim was effehte.#-,'. .

fey Brink (If#2) disoowred that a water extract of sterile

yeast added in aiali amounts to sugar medium stimulated pollen grain '

gerpinatioBo Brink (192i|,S:;#l and #S) 'reported that he detected an

increase in total gemiaatlom percentage fey the addition of yeast to

an artificial meiiamt,

Baaliker ■ (1936). was led t©: feeli®T@ that the yeast used by '

■„ • ■‘i- f't , - - ^ ■ " " • Brink ©obtained Titamin B^-o Sntoher (1918.) ' demonstrated the presen©®

of Titamim Bj, in maise polleno Me proved that with .the addition of

fitamia,B^ in ;a, sugar 'solmtioa^ .ihe peUen of. Garioa fueroifolia. showed :

a sipiifieaat inereas®; im total germination percentage ever the control©

■ ' lost pollen grains germinate successfully' in'- sugar solutions©

Suerose has. largely^ been usedg but lactose^ dextrose^ > rhamos®8 ..raffia , 13 nese9 aaad galaei®s@ gemwally give g@@d [email protected] #ile fra@t©s@a ma$m©s@9 aa4 maam-tol are reported as msatisfaeterjo Johri sad

Vasil (1961) and Bair and Looms (Igi&l) stated that amis® pollen geminated poorly on frmetes® and progressively' better m ^.f®erin®s y.m@0s®$ and sm©r®s@ m@®d at equivalent molalities^

Sine® the beginning of this @©atary9 there have been two general se&ools @f thenght §@n©@raiag the qm®sti©n9 ^B© pollen tabes ' growing in vivo and, im vitro mtiMse externally smpplied nutrientsfM

The te© main views eon© era the endogenous or the exogenous utilisation

.t. - of nutrientso & vitro means that the exogenous nutrients are added to the medium te stimulate pollm gemiaatioa9 whereas^ da Vive is used to indieat® that eessgenoms mntriemts are not required ©r supplied f©r pollen germiaatioBo Aeeordiag to Brink (192ks #3)s the length @f the pollen tube to vitro is ©onsiderably shorter than to viv@g

Inthosy and larlan (1920) believe that'externally supplied, sugars, have only am osmile role and are not utilized by the grain for any nmtri= ■ tiomal purposes® Martin (1913) is in eomplete agreement wi # ^nt&oay and Harlan® ' Brink (192h$ #3 and #%) and 06Kelly (1955) believe that apart from having am ©saoti© r©l®9 the externally supplied sugars to the medium (or .to the style) definitely serve as a nutrient material for the growtog tube®® As reported by Brink (192% #3 and 0k) when, a medium with laetose sugar is us«d9 there is a profuse bursting of pollen grains and often a® germination at all® Addicott (19fe3) ©oa« eluded that pollen @ay supply ma^ 1 of its requirements from its mm • reserve® He pdstti#t#d% that ::pheh##ma ©f germtoattom and tube growth are :#@t neeessp'ily related® m T E B B I S # B ffiTHOBS

• ' The praeti^al r'alae ef the ariifieial geradnatioa '©£ sosghm poUea is. .ia its :m$ei .ia; deterzniaing the physiologieal and eavirea® mental emditioas aeeessary for seed seto Alsos onee these eohditieas for normal pollen germnatioa are specifieds shipment of sorghum . pollen under' controlled Conditions may he possible@ Easy pollen e%« ehange mi l faeilitate - eressing and will save time as i?eU as money im a hreedimg pregram@ ;

Eomree of Pollea '

.A,t the beginning ©f this stta%s pollen need was collected from

Sorghum vmlgare .plants that were grown -im. the Plant ■ Breeding green® homse during the winter of. 1960elf6lo; ISiea the growing season started in the. very early summer ^ sorghum fields'located in Avra Valley^ west

©f Tnesons ' Aria©Ba3 beeame the soweeef pollen necessary for this study0 ' laters the' majority @f the pollen was- oollected,from different

TOrieties and hybrids.: grown. during the summer of 1961 at .Th©; :Bniversity of. Arizona Gaupbell Avenue F a m d Tues@ns Arizonao

The varieties used for this study included Plainsman^ Martin#.

Combine Kafir 60g Radian ^ Combine 70?8a BB®38# Spur Eeterita.# and

Tracy* -Pour commercially' adapted .%hrids grown-in the southwest were . ' also mseds P8®6lQ^ Amak ;B®10s .Amk B=>123 and BeKalb

Test' Conditions:

Trials in germination of pollen were conducted' St several places 2' • (1) in the head house: adjacent to. the Plant ’Breeding green®' (2) in. the Botany laboratory9 and: (3) at the Campbell Avenue

Farm, The early, morning temperatures in the head house were recorded

- : . • 6 ; ■ . ' ' , ' ■ • . ' ' ' ' ; ' ' " . ■ between 80 and 90° whereas? the relative humidities .were record=

ed between 70 and 80 per cento The temperature in the Botany

laboratory was maintained at 70° F<, and the relative humidity was

approximately 20 per cento Pollen studies were conducted under pre­

vailing climatic conditions at The University of Arizona Campbell

Avenue Parmo ’ ' ■■ ■ ■ -

Stainability - , . -.

A stainability test was conducted to check the percentage of

good and bad pollen grains from the time of anthesis until pollen

grains ceased to dehisce from the anthers<> The presence of starch

within a tissue can be readily, detected by its reaction to form the

intensely blue starch-iodine complex; consequently5, pollen grains that

contain starch will stain a deep blue-black,, whereas 9 other pollen

grains will be opaque <> This stainability test is used on the assumption

that stained pollen grains measures starch content9 which is correlated

with the viability, of pollen o. Combine Kafir 60 was used for this

particular study0 PoUen was dusted from sorghum heads on each of

four slides every hour6 Pollen was collected at 3sOQ9 SzQQ? 7t00s

8i00s 9:009 and 10:00 Ao Ho Anthesis generally started between ^sOO

and 6:00 Ao Mo for the variety under consideration0 This incidentally^

is in accordance with data published by. Stephens, and Quinby (193I4) \

who reported' that anthesis. in ;Sorghum vulgare is, dependent. upon the

variety itself 9 and that temperature and humidity is directly correlated

to .anthesis in any variety0 This study was observed from August 109 1961 to August. 26s 1961©. : Environmental eenditions of humidity and

temperatir® were reoorded on a hygrothemograph in order to eorrelate

the;f a©tor with stainaMlitys Slides seated with pollen were taken

t# the laboratory and. each slide treated with a $0 per' cent iodine :

soluti©n0 drains were observed ■under 1© X magnifieation» Counts of

stained and clear pollen grains on each slide were made of at least

seye®/Bieroseopi© fields,®,, . ’ , . -

kempts at Germination and Beseription of Methods

A Series of tests have .been conducted to devise a method

suitable'to induce artificial gemination of Sorghum vulgare pollen^ '3 These tests led to the simultaneous study of some environmental factors

that influence pollen germinatione The environmental factors studied

were humidity5, temperatures'1 light9 and age of pollen grains in reject

to germination0 ' V-' - . ; -t.. / ■

Antoesis and Pollination

In the first trials to artificially germinate pollen-in the

. laboratory«, heads from eu3jtts were out in the late afternoon and brou^at

to the, head house© It was observed that pollen dehisced, the following

morning but would not dehisce thereafter0 A laboratory method was .

then devised to'maintain a continual flow of pollen as long as anthesis

oceurs. in a sorghum head© following the suggestion of Stephens and.- .

Qoinby (193k) that anthesis starts in the top one^third of toe head and

occurs for approximately seven days in a sor^um heada plants were

selected in the late afternoon' where; anthesis had started in the top -

©ne^toird of the head©. The heads weyeeui above the top node of the

stalk © The stems were immediately placed in, a bucket of water and- moved t© the. head hemseo Individmal tesds were later' t.EaBsfex,£,ed from the bucket te a shallow eeatalner ©f water g., ’The eentainer ■ - . •• .. , _ s: ■ ' - ' measured fe w inehes in depths ©ne and ome°half feet l©ng2 and one. foot iald©e A weight was submerged la the container of water and s© arranged-as to hold the ent stems beneath the water0 Bj placing the stems in a nearly herieental position the entire portion of the head

.will be ©ntside the edge of the ;©©Btain@r©,,' Fo U o m n g this procedure - of ^keeping'the . heads in a horizontal position's' with the stems smb» mer'ged5 the pollen will dehisce for approximately five dayso T© maintain heads in a eondition to produce pollen^ water should be changed every dayo Anthesis of selected heads oeeurred in the head house at the conditions previously mentioned (8O°«=.;90e F 0 and 70«8@

. per cent relative hiW,dity) .beWeen # : # and 7##; A* Ho T© eoXXeet

' ' : • ' " ' ■ l' ‘ ‘ : a. ' , - ' , - - . . ' ■’/ ' ' pollen On sUdesV the heads should be gently, tapped only .oncej other® ms©s if repeated.more than ©noeg immature pollen grains will fall#;

Germination Under Hatural Field Conditions

.. . 1 Sorghum pollen was germinated in petri .dishes under prevailing

■ environmental factors of temperature ;'and' hwri.ditya Humber three ' ' filter paper was- plaeed in the bottom of petri dish@ss one or two drops of distilled water, was added to the inside of the dish to obtain a high humidity in the. eldsed containero Pollen grains were -dusted on - slides by gently tapping sorghum heads in tidiioh anthesis. ®@eurss thus liberating the pd.ll©B.o A pollen coated slide was quickly enclosed in a petri dish and, plaeed in sunlight or partial sunlight where temperature . ■ ■ ' Vi' ■' f ; ' was recorded .at 8$' F@ \ . ' ■ ■ - . ' . / ; 18 ' ;

Geg’mlaatlon gnder G-reenhomse Conditioaa. ; , ■

,' S®ryam p©M.@B- was germlnaied. 1h 5©tei: disties rader greens . : house conditions o Number three: filter paper if as placed in the bottom of petri dishes^ two or three drops of distilled water were added-: to the inside of the dish.to obtains high humidity in the closed c©n= tainer®, Pollen=>eoated slides were -enclosed in, the dishes® Petri dishes- were placed in direct sunlight- and temperature was recorded at

85 Fa The temperature under greenhouse conditions does not. fluctuate as much as- temperature under field conditions^ thereforei,- germination studies were conducted in the greenhouse^ ■ . , -

To germinate pollen - during the fall months in the laboratory s when: the. early morning temperature was below 60° or 70® blue blotter paper was placed in the bottom- of petri dishes to increase the temperature of the enclosed, containers Blue blotter paper- absorbing light raysj increases -the temperature in- -the enclosed container* *. - -. .

Chemical Solutions--used on' Pollen'Grains : -

: sk was: suggested by Artschwager and McGuire (19ii-9)s an agar solution of lo5 per cent was used to germinate sorghum pollen® ' ;

Different concentrations of sugar .and salt solutions-were made . separately and. stored in 25 ml®, flasks each® - The following percent­ ages of sucrose solutions.were useds ,1* 1o55 2S 2@5S 3.s hs, 5S 10$ 15 =

3QS and is.0 per cent®. Various solutions of salt .(sodium chloride)• were used:. lg 2S 3y ks 851: and 10 per cento Sugar was used to supply : , a source of energy for .pollen grains^ whereas sodium chloride was used

• - \ - ■ ; - -■ ' • .. - • .. . ' in order to determine ■ the; osmotic effect on pollen, grains Brink "

(192lis IE) suggested proteins were important in pollen germination and’ that gelatim. /#8 a g©©d souree. o£ protein0 Consequentlyj, toenty"

fire b 1» each of 1« i®5@ and 2 per, eeni gelatlm sototions were made©

A eomplete' nutrient solution. Has obtained from the Botany Departmento

The varioms solmtione msed^ separately; and in e©mbination>- 'appear in

Table 10 - " : . . ■:■■■

Plant 'detracts \ ' \ • •,

• Material, tised for plant erbraet was taken from three separate

portions ©f the flower plus extract taken from a mature flag leaf = By

: .inserting a mixture into a © entrifmge for .a definite amount of times : _•

, different components of the pistil and leaf extract will separate out

according to their 1 respective. molecular weight So ' Therefore-, one ' .

, - - ■ ' . ' ' * : hundred pistils were extracted from individual florets x-ixere anthesis.

had Just started^ one hundred pistils were extracted from florets where

anthesis had ©empleted’ it® eyele§. twenty pistils were extracted, from

flowers with immature anthers,, and.mesophyll ©ells from the flag leafa

sompilsed the constituents used as plant extracts0 Each of these

portions of the- head and the mesophyll cells were maseerated separate™

ly and fife mis' of distilled water added td each* Each portion was

placed in a centrifuge and spun at 2g230 times the force of gravity

foy three-minutes0 '■ These- various; solutions of plant. extracts were ■ . . :

used in agaro Sonfeinations and different percentages of sugar plus

salt in agar| sugar plus salt plus proteins in agarf and . sugar5 plus

saltg plus proteins^ plus plant extract in agar were, used, in an ...

attempt to germinate sorghum pollens

V isually.-©ne liter -of agar solution was mad© in the morning and

poured into a series of petri dishes while warm® The solution or - 20

TAB2E 1© Goacaafeatioas e£ Sugar and Salt.^ Salt aad Gelatin^ amd Plaa* Srfetoeto^ .iiHsena 19#lo

PerieSage . ,. s. . §©a@eatrati©a s Pag'seatage G eaeeatratloa ,©f Sug a r' n o - - -V® ' S - 1 # *Xi -- I- X;:.8••X: I >X = $ ;-X.>8 -•;X,';g.--,-X.; • ; . - ,1,., ■ - . 8 X- 8 ,x.£ • .2,%' g , , 2 ,% ,2 X. 8 ’X.8 • 2 . .'.x. f . f .2.. . ■ 8 ,X. 8 -X .8 ••x. .s,%.s . . 8 .X. 8X. 8 ■• X -8 2 .-X.8 • -3- -, ... rTx/TsL 8 . . g :±'. g. - 8 X ■ 8\% /2 / 2 ,.% ,2\ X.8 - ,3C, , , ■k: - •• f-r-'X - 8 .x. 8 . .2 % g % S X 8 % .2 - X.8. x.s. -X. 8 . . ■ 8",x • 8 -X- 8 : .2 -X - 2 •L-%/_,2 % ;2•x. 8 •X.8 X - 8. ■X-B . . 8 X 8 X. 8 • .8X8 .2 .%.& X & X.8 ..% .2 .X.8 " V -.. .10...... , 8 -X .8 .X. 8 . .. 8 X - 8 ■ . 8 X - 8 X 8 - 2 ,.% .2 ,% - - -»■ i Gomeeatratioa•i &£ G elatlB s

J...... : . g -X- ° , % ■ $ . -3C, g X S .3E. ■ S .% & 'x.% ■ X- 8 X-S X 8 . .X- -.lo57" ,XS -X ,8 ■ -X . - o_X, g ■ g X . S. X 8 X - 8 : - £ . -x. ■ - -

8 - X . g .8 X. g - X S X .8 .X-8 - -X 8 - X . 8_ -X 8 . -X-

. 8 . Elaat Ixtraet

I* a tu r @ . . . g . ...8 . . g . . g . g. . . g. .% . .8 - . g-' , . g: P is t ils 8. X S. X 8 8 X 8 X 8: X 8: X 8 X S X 8 X Hates1® — . 8 . .8 g . 8 . . . 8 .8 . .8 . . .8 . P is t ils S X 8 8 X 8 X 8 X 8 X 8 X 8 X 8 X X 8 X

■ '£> ^ • O * s ^ O -• ^ ^ » Q • • • O - - v O .r * - O - ■ ' i3 - • - O — - • ' O f • ' - O « - * • _ Extract "8 X § • X 8_X 8 X. 8 X .8 X i X 8 X 8 X 8 X 8 X

0®m©«trati©a % , gG elatia 8 . . i . .2 , . . 1 .....2 ,3r 2 2 . , 8. 0%' 2 .2 /% 8 % '2 . Z .2 \ Z 2- . z . ., / ,.X, .2 ," n T T x x 2- / %T ,2.,% i: "T 2, Z \2 , X.8. Z 8 .z , ., , 2 , ., 2 .. . 8 X..2 §. TT.% 2 , ;g . % : 2 8 . X.8. X. 8 . z 2, z , ,, ...2. : 2 , .. 1 , . .. .8 X <2 8. , 8- X 2. , 2 % , Z , 2 . X " 8 z :. -X.. - . .2, ,2 .. .lo F T 2 .2 -2 . X -. .2 .% s , %; 8. X ;■* ,3C 2 . X : 8 - Z ,2 , z 2 . x . .. , .2. .2 , , ,2 - ■ . "2 x.T , .2 2 - . g .Z ,. 2 .z" 2 -Z^2 .Z 2 z 2 . z . ,. TX ;s . 2 .2 % .2 , 2 .% l . ,2 s 8 - X - 8 .Z,2 ;z s. z . - -3-v2 . .2 ,2 % 2 .8 X ? • 2 .%.. 2 2 . ,2- .X-8 , z 8 -z 3 - -2 -.. .2 ... . ."t-X . B .2' , 2 .% 2 . . 8: X ■■ S S - z .g . Z.2 ^% & .Z. n --2 , > - » * 8 -X■ %.3C. 2" ,s X 2 . . 2 2 z 8 - X . 8 • Z : 8 . z 2 .3ETTT i& -2 -trioS T: 2 % JL X »L. . 2 • % 2 :i l 2 : Z .2 . Z 2 ^ X 2 . z / . ^ , 2 , .2 .. . .? %. t X, 2 , 2 3C 2 . 2: .% • ?. % 2 .-3E.3, X.S. z 2 :z , .. . .6. .2 V..ITT. 2: ± .2 2. . ,.2 % 2 . .2 2 z . g .Z /2 .z , 2 , z z ... ■H * . .6.. - 2 . - ic i r : .8 2 2 2 z . .2 % 2 z . 2 i Z 2 X .8 . z s’-z , .. , ,0 .. . .2 . . . .2-3C.-g. %[ . 2 -% 0 - .2 2 z : . 2 .Z Z ;s\ z ' i -z , ..

igm ifies ©©mfeiaatioaS-.^ad tke apprepriat© @©aeentsati©as msed soliations @f smgaE'g salta plast extraet, gsliS£% and complete

SHtrieBt medimn were added i© the. warm agar and allowed to solidify®

- • .Polleii was them dusted ■ om ea©h agar plate and ©aelssed im- a petfi :

disk and these were stored ra&er e oatrolled room, teisperat'^'es at 70° F 0

' ifter an k©mr«, ike plates were examined tmder s MaoeuXar microscope

.. for ®©r^^. pollen' genainatioa and all plates were observed each day

.thereafter matil the growth ©f "bacteria and ftmgi made ©bserwatioms

irapossiblee

Ghem3.cals in Agar . . • '

Agar solutions ■were miiced at might and Tarieras eone ©mirations

of smgar j pr©teiBss:. plant extracts.^ m d ^ther chemicals were added® :.

■ Two slides each were dipped in eaeh solution and placed in a eomtainer

■ :i© designed to maimtaim a reasonable high per C emt of humidity0 The

. eomtainer and contents were stored at room temperatures® The follow-

img morning the coated slides • were taken to the field® With the

:. oeeurrmee ©f aathesis is Combine Kafir 6§s:. shosea. for this particular .

studys pollen grains were dusted on each of the two slides for each'

. - of the different - ebnoentrations ©rf Maetures used and taken to the

laboratory for counts® A : microscope was used to determine the percent- . ;

• age of germination by mkim.g counts every half hour® The study was

. repeated' using: agar in petri dishes instead of ;agar, coated slides®

. Paraffin Studies - . 'v -

. ■ Paraffin, ©dated slides were taken te the field in a container

and pollen dusted on them and then these slides were taken to the

. laboratory for count® Pollen was also - collected on paraffin slides

: from sorgl^m- heads,maintained ,# the head house and in 'the greenhouse® '■ :" - ‘ \ - The paraffin eeated slides eontainiBg the pollen were als@ studied

. ' ' . ' ■ ' ' . in petal dishes» Two drop's ©f distilled water were added t© each

petri dish: t@ ©hiaia a ; high per eent ©f r elatiir© himidity0 The

petri dishes were^eevered and plieed .in direct smLight where ihe

temperature was recorded at §5° Ft Slides were examined f©r pollen

g®minati©B. after thirty giinntes ©£ this treatments

If feet ©f Light ©n Pollen Germimatiem in rive -

' The physiolegieal effect ©£ light and temperature ©n pollen

' gemimatlen rad. pollen tube grPsth cam he determined by the use of &

chamber that controls temperature and light© T© ©Main various ,

relative timiditiesg sms,11 indiiri-dual chambers were so designed as

t© maintain a given humidity and these in turn placed in the

temperature' and - light controlled chamber = Spencer (1926) * © devised - :• . 1 1 - 1 1 ■" ■" , . 1 v ' ^ ■ . 1 , the following method states that by properly selecting the salt t©

be,useds aaay/described degree of. humidity ©an be secured and controlled^

This will maintain a constant humidity at a given temperature wipin' a

confined;spaee.o Table 2 shows the per cent humidity,at a given /.

temperature within a closed space when an excess @£ the substance in®'

dieated is in contact m t h a saturated aqueous ■ solution ©f the given

solid phase©. The salts in relation t© a specific humidity rad specific

temperature were taken from the Physics and ChCTiistry Handbook (i960)

and from the work fey Spencer (1926)' recorded in the IhteraationaX

eritical.Tables'^ • . - . .

Thirteen individual humidity chambers were used which consist®

. . ' , , . ’ ...... ■ ■ ed ©f two t^ies ©f deep frees® cartons ®. a polyethylene carton that. '

admits certain light rays and a clear plastic deep freeze. ear ton 0 . A £3

fABIE 2o' Humidity .at 'SiWA. Tmperatnre ■ Witiaim a Closed .l--.- .: Space We n an Exeess-;®f the.Substanee Indieated is is ■ SoBtaot m t M a Saimsated Ataems -Solmtion ef the #ivm ■ ;s©3M lhasei,* ' / ■' :: ' :: ' sV...... :. . ..- ...... Solid fhas© $ Temperature ®6o ? Per Cent Humidity i . %

% o4 igt- Sio® ^ Mg q>6 Hg@" 2&o5 52 e©

£5<>® ■ 71c2

1H|: EL - : ■ 308# \ . ?7®5

ci . . i5»# '.. ■ 79o3

m k ■ / ■ • ’ -3#c0 - . .' Slol laiig **"1^.': ■ Se5': ' 88 o©

% % / - ' 3©o6 ■••■■ 9So9 / : £5e0, ' f.3o®

"I960 Chemistry, ahi .fhjsies. laAdhoofe^ pp© ffe9^«25@©o ' Bomber /five jptitoer sieppes .1® ! pissed In the hottem @f each ehamfees to: support the slide.'above the s a lt soly.tionss and the necessary salt. /’

added to each® .' S is tiile d water "was added m til each salt, seluticm was im a'saturated c oaditiOB®;-

■ .:-,.$he eontrol chamber was maimtaiaed.at.a temperature desired for

each series of tests.*: A H individual hmiidity chambers were placed in the control chamber and'preheated for at least 2% hours before use® A

. hygrothemograph was placed in the large chamber to record, temper at we Q

This experiment was e endue ted. in" three phases each for . ' ' '

temperature and light®. The three levels of temperature used were

28®r,. and. 3©® Each temperature was maintained for each of three

treatments of lightj light source from fluorescent light# a Watt .

. lamp to supply infra red light rays3 and one germination trial was made

in the absehee of light® : " i . ■■ ■ ' •/"

Pollen grains, were dusted from, mature heads onto glass slides

and these placed on top of the rubber stopper inside'each individual

humidity chamber» These chambers were covered .and placed in the

temperature and light controlled chamber o Souats of total germination were mad® under a 10 X microscope at apprerimtely thirty minute

intervals®:, . ■ '- ■ ' . \

Effect of Eight on Pollen. Germination in the Field

A study was conducted to determine, the effect of light on pollen

germination/on stigmas® Eight varieties were selected for their

commercial value in the southwest and combination purposes t© afford- more diversity of material® Varieties and hybrids selected for this '

. study 'were ?; lediah.s ; Hegari> DB=38^ Martin^, B® Kalb 1=55s A m k R»10s • SB .6l©«, and Combine 7Q?8e " Heads were, selected that would, be in

anthesis within six or seven days and four heads were bagged for '

eaeh variety^- Two with normal sorghum pollinating hags.in which / .

pollination is expected to occur normally9 and two with black bagss

"t© e&eludie limits ■ The black bags were twelve inches : in length and.

six inches wide#' They were made out of black construction papers

approximately one^eighth inch thicks These bags were folded and

the black bags in an attempt to keep them at temperatures comparable

a normal pollinating bag and inside the black bag covered with the

corn pollinating bag® Seeds from normal heads and heads covered with

black bags were harvested from each variety and weighed®

The Pollen Grains Exposed to Air

,4 study.,was conducted to determine the effect of viability of

sorghum head on each slide and exposed to air. for ones twos threes

. fourg fiyOg.:and six minutesQ Each slide after being exposed to -air:■' •

for a certain period of time was placed in a petri dish and subjected

to germination conditions® Semination counts were made after ten

minutes® The graph on Page shows the per cent pollen germination

for each minute correlated with the total per cent shrunken pollen .RESULTS

The artificial genninatioB of plant pollen has met m t h medioere7 smceesso . Pollen helongimg to the Brsmaseae .family has been most iiffienlt to germinate due to the extremely delicate mature of the pollen grains» Attempts to germinate pollen and find, the optiimm

- environmental eomditions meeessary for germination has proven to be a laborious task © Sven nows additional studies are needed to more accurately determine the effect' of environment on pollen gmdnation and subsequent pollen tube growths

Stainability test '

■ ; ■' Close observation ef the Combine Kafir 60 variety iadieates that little; :#nthesl%-' oeeurs before 5 s 3® £ • ;Ho T M s was evident by the amount of pollen collected 'at 5 §3®s 6iOQs b$S©> and ?i®0 Ac Mo

The peak shedding - ©f. Combine Kafir 6© pollen usually occurred between ls®0 and 7:0© A« E» The flow of pollen begins to decrease after

. SilC Ao Me ”and 1® tismallly exhausted by ’a®s3®-'A©. Mo The effect of

'environmental factors ©n daily pollen shedding^ as reported fey Jones

"and Br e w (1951) g was Verified fey variation, ©f. the daily peak

.pollination period ©f' Ges^iae. Kafir 6® sorghuma .

The highest percentage of stained pollen grains occurred at

. l©s:@©.Ao; Mo The average per cent of stainable pollen grains' for each of the eight days the study was conducted' is listed in Table 3® Tables h and 5 list the humidity and' temp®raiur;e for the days the study Was conducted® ' Figure 1 graphically- @©mpare:|':.tb@. per -^gsb of stainable

■■■'vV;. polXeB grainss the per cent relative humidity^ and the temperature

(@Ze') for the days the study was conducted© Figure 2 shows the per

cent stainable pollen grains for each of five selected days during

the study© S day hy day analysis relationship of the correlation of per cent stainahle pollen grains, per cent relative humidity, and w A » , . - r . . y . . f - » « • - - ' — ' “V 1 —*« T ' 'W- ^ -a— • ‘ “ *- - *■ v * * ^ ' temperature (®Fo)' is shown on an hourly basis for each ©f the eight

days the test was conducted© Comparisons appear in Figures 3S Us 5s

6a 7S 8 s 9 s and 10G It is of interest to note that the highest per

cent of stainable pollen grains usually occurs - * m the decrease in humidity and the. increase in temperature were approximately equal

Studies, with Solutions '

Studies .conducted in the laboratory to artificially germinate

Sorghum vulgare pollen were made using.' water5. solutions of ■ sugar plus - waters and solutions containing other nutrient substances© Pollen

grains failed to germinate in the various tests conducted©. In view

of the negative resultsa. only the behavior of pollen grains in sugar ,

solutions will he r eported because physical changes were noted that were interesting and appeared to be related to germination of the pollen grains © The results observed were? (X) high concentrations

of sugar resulted in plasmolysis of the pollen grains* (2) low eon»

eeritrations of sugar; solution resulted in bursting of pollen grains5. whereaSj, immature pollen grains increased in sizes but did not bursty

and (3) pollen grains, placed in 15 to 2© per cent sugar solutions

formed small knobs by the protrusion of the cell content through the pore from which the, pollen tube emerges when germination takes place©.

These knobs reached various lengths and displayed various shapes® but do not grow furtheri, which boeame a standard of pollen grains that

. would, not germinatee.

Trials were made to artificially germinate sorghum pollen .

following the in vitro technique by using various extracts from

: sorghum flwers antithe various nutrient solutions in. agar as' listed

in Table howevers - aH trials resulted in failureo. Sueoessful

pollen germination ©eeurred- when the' approach- was changed and : ■

germination was attempted to Vivo "by eoating slides with pollen and :

• introducing them into a petri dish supplied with moisture^

These ohservatioms'led to the eonelusion that germination was

dependent upon certain emrironmeatal factors«, and the laboratory .

eontrol of mrironmental conditions was absolutely essential for

artificial germination @ . 7

T A M S 3o ■ $ W Gent of; Staia&M,© - Bellen Grains for 8 'Bay Period© Tuesons .Arizona ° Angasts 196l0 , ; . „ .

Tim® Bat®' ' • sAverage .s:Bj25”gPer Gent

O V V ' »«■ < * -r .d- ,-* •» ^ -- ■• 8 * .- - •*’ ►* —*' H £> ,*■ ■« i • r * ►© >■ .y \ *• " ..A . V O Jf* =• .» * ' & “ . - ^ y r 0 >■* r ■*- ■» ' (• O 6&# Ao Mo ;? gko8. g 9 5 : 9&o2 % #^8 $ ® «> : 95o6 % 95©8 i:"9;8o2 t 95° 19 : ‘; ;s I 7'$0Gl- $&* •'•'$’■ '8 c=» fe S =» m • $ '$$&&■ 4’ •^3^" :-8 ; 9606"'8 9208 8 97®3 ’:$™9'5<$’G5 ■■ / . 8 ■ 8 ■ .8 : 8 8 ■ , 8 8 8‘ . 8 8i©0 io.So 8 93©G I 92o8 f 97.ol 8 96G1 : 9k.9 8 95=9 ? 9ko5 § 97©2 8 95*19 ■ ' ... . !. ' . ..? T . ' * % . ? ... ! ,* f ; •• f z®@ M Mi't 9ko6 g = <» § 97®1 t » 9&ak 8 95o9 » 96a8 8 96#l6 8 8 8 8 S 1 8 8 $ 8 IGsGO I© Mo' s 99*2 i 95*1 f 96.08 s 98©5 i 'a “ % 98^5 8 98©2 s 98©3 ? 97eU .8 8 8 8 ' 8 8 8 ' 8 8 TABXE I# Per Gent' 'Htaroldliy in Sorghm Field at Gan^bell' ■ STeame fame fues#Ba Arizeaa = Arngmstg 196lo

' § .$7gB7:PeF Seat

. SiGG Ao Mo$ 93 s 93 s; 93 | k3 t 83 s 93 s 93 s 91o5? ' ° s ‘J. ! g ! S ■ i f 7300 .Ao Mo i 7© ' s 93 - ! ■ 65 s - k2 §' 67 | 93 | 93 8 93 g: 82o©0 ■ 8 s s ? - . ? g - ? ; ! S 8 800 Ao Mo o- s ; 66 30 36 56; . 8 93 8k § 70 t; 6ko86 ■; ' ! r ■ ; '! s s s ? ' ' ?: ■S'. ! S 9800 , Ao Mo 3 W k9 s 2:3 s. ,32. , 8? k6 60 g 5k g k8 s k6o57.: ! .■ : • ' 8 s i ' f •:' ‘ : ■ ■,? i 1 -■ ? ; ?: 10300 Ao Mo a 35 ! 37 . f . 20 ? 128 | 38 k5 t a g: 38 t 36o77 • ; . . 8 ; s t ... ' ' , 1', ; § ■ -8 s g i

- .

TABXE 3© Temperatmre (eF) in Serghtm. Field at Gsapbell AYezme Farm© ...a ... • ?mesoBs. Ari$tBa,,.« AmgBgt$ 1961 o _' .

Time i Bate; ^Average _____ :: TijPer Gemt

/ ^ ^ * -" •' <■ v / ^ - -> ^ ^ V jr - - O* r * - * * -* O -# y y a» -• O x , y * S t O » ' y v ■»■* •” ^ r - >■* y ^ 1 6?O0 Ao .Mol 68 i 71. s 71 * ; 8© s . 'f6 % 7® i 71, s 69 i"72»l0 ' ' ' ■ 8 ' ■' ' . 5 ' 8 : - ' § ' ’ ■ .8 ' g $■ g ' 7800 A® M<>f 7 2 s 70 ! 70 i 8 2 I 79' ? 70. f' 72 s -69 & 73*00 s,' , . , 8 . . : . g- • ' ? V : i • , , 8 ■■■ ?: : f " f ■ ■ : ' 8300 Ai Mo 8 78 3 75 I 83" - g: St s 72 £ 76~ 1 ?3 £ 78©12 8 8 1 8 ■ . 8 ■ 8 8 8 ,. 8'" ' ' 9800 s lei ' 8k 8 Sh 8 - 89': > ■ 89; i 87 r . 77 ! 80 g 82 : SkoQQ ' 8 8 8 , 8 §■ 8 8 8 -I l® i© 0lo Mol 88 8 88 g: .92;' i 91..i 91 i . 81 a 87 i 86 f 88o©0 ■ 8 : 3 8 8 .: 8 ' 8 3 - S &' 31 Temperature

6 A.M. 7 A.M. 8 A.M. 9 A.M. 10 A.M. Time in Hours

FIGURE 1* Showing relationship (average) between stainable pollen, relative humidity, and temperature for eight days, Tucson, Arizona - July, 1961, 32

100 * •

July 18

July 22

July 2h

July 2^ July 26

Time in Hours

FIGURE 2, Per cent stainable pollen grains for each of five days. Tucson, Arizona - July, 1961. Per cent FIGURES 3 AND U# Showing daily relationship between stainable pollen grains, relative humidity, relative grains, pollen stainable between relationship daily Showing U# 3 AND FIGURES n eprtr ne fedcniin, usn Arizona. Tucson, conditions, field under temperature and o 5 E-f u I to a)

Temperature FIGURE 5 August 21f 1961 FIGUREj 6

e Humidity 0) Humidity u Temperature Temperature I Stainable u Stainable Pollen Pollen 0) E-t Temperature

Time in Hours (A.M.) Time in Hours (A.M.)

FIGURES 5 AND 6. Showing daily relationship between stainable pollen grains, relative humidity, and temperature under field conditions. Tucson, Arizona.

w f=- FIGURE 8. August 2k, 1961. FIGUR£j 7 ■»■■■ ■« ---;---

90 90

80 80

70 I 70 o

Uo Humidity h0 ‘Temperature 30 30 ‘Stainable Pollen Stainable Pollen

7 8 9 Time in Hours Time in Hours

FIGURES 7 AND 8, Showing daily relationship between stainable pollen grains, relative humidity, and temperature under field conditions. Tucson, Arizona.

% FIGURE 9 FIGURE 10. August 26, 1961.

100

90 Temperature

$0 Humidity Humidity Temperature Temperature Uo Stainable Pollen Stainable Pollen

Time in Hours Time in Hours

FIGURES 9 AND 10. Showing daily relationship between stainable pollen grains, relative humidity, and temperature under field conditions# Tucson, Arizona# 3?

Pollen Germination

Tli© morphological characteristics of pollen grains in the

Gramjaeae family have been described by Erdtman (19k3 and 1952) and

Wedehomse (19595 o The growth of pollen tubes in the Gramineae family

- ■ - - J. . . . - ' ...... has mot been deseribed aoGor&Wg to the literature reviewed*

Repeated observations Indicate that sorghm pollen germinates in the follouiag manner a Apparently^, idien some specific limit of relative humiditys temperature^ and age of pollen reaches a threshold^ the grains germinate^ A single well-defined pore can be observed in the pollen grain,, and different stages concerning the development of the pore will be evident

■ ■ . *:• region of a mature pollen grain and then the pore becomes more and more evident until a supposedly mature pore is developed (Plate 1)0

As the apical portion of the pblles.. tube * grows through the pore ±m cress section^ it appears to grow anticlinal to the intine and erine0

The germinating pollen tube ceases growth in an anticlinal direction as soon as it grows beyond the erine$ then the small germinating tube grows perielimal to the exlae for a very short distance (Plates 2 and

3) o Apparentlys the portion of the germinating pollen tube that is growing perielinal to the ezine stops growing^ and some portion eon® times to grow longitudali^r in a random direction* but not perielinal to the ezine© At this time the germinating pollen tube (Plate k) in­ creases in diameter* and are observed to be at least one and ene-half times as long as the diameter of the grain itselfs

The conditions in the field were duplicated as nearly as possible in the laboratory© Pollen was dusted on a glass slide and

. ™ " . 38

PLATE 1. (Sorghum vulgare) Showing (1) intine, (2) exine, (3) mature pore, and (U) the start of pollen tube growth. X 1000. PLATE 3

PLATE It. Mature pollen tube. X $00 " . , , . - : : , ' placed In a moist petri dish and gerrmiBaiion occurred within ten

minuteso Temperature and relative: humidity that gave the best ■

. germination was determined to be 80o«85o F®. and 8?=»92. per cent

. respeetivelsro Ihen the relative humidity within the closed petri dish

exceeded the critical limits ®f humidity required for pollen germina*

tion. a small- percentage of bur sting-was observed®

Humidity Ohamber Results .

. - HoHea geminated best when fee humidity chamber was maintained

at 92 per cent relative humidity and- at 859 F c ■ Pollen grains placed

in hxmidity chambers that were calibrated for relative humidities from

ten to thirty per cent tended to remain the same in si^e^ shape> ete*,

with the exception of partial shrinkagej, which indicated a small loss

of moisture® In high humidities (50s 6©s and 70 per. cent)s immature

pollen grains swelled to normal’size but f ailed to- germinateQ At the

humidity level in which germination © eeurreda a small percentage of

mature pollen grains bwsSedo A list of salts and respective

humidities produced msixig them' within a . closed chamber at a. constant

temperature was given in Table 2® ; .

Light Control Chamber • 1 ' : ‘ ' ' Pollen grains dusted on; slides, and placed in the series of,

individual humidity chambers and incubated at F® failed to

initiate germination when ;a> light source was absent®' /When observed, at

ten minute intervals in the. absence of light. knob formation was ■

evident om pollm ezppeed to relative humidities above per

eentj howevers where pollen grains remained in fee humidity chambers

for two hours5 profuse bursting of grains occurred if fee relative htmldliy was aiDoye ?0 pep sente ■ Ihea light was supplied fresi a -

fluorescent souree^ no germination oeeurred bmt with the addition of

light supplied from a 75 Watt incandescent Xamps sporadic germimation

occurred when the relative humidity was 92 per cent©

Light Controlled Field Study

That light had an effect bn pollaa germination and subsequent

pollen tube growth being thus determined^ the effect of light on growth

.of pollen ©n stigma tic tissue was next attempted© The weight of seed

produced on normal heads compared to the weight of. seed produced on

heads in which light was excluded was compared on a percentage basis©

The weight of seeds produced on varieties Hedlan and BD=38 grom under

black bags was higher than those under normal ■ pollinating bags» For

all: varieties and hybrids studied^ with the exception of Bedlam and

"DD”38s tlie weight of seed harvested from heads grown under the black

bags as compared to normal bags ranged from ij.0©6 t© 8l08 per cent©,

The weight ©f seeds under: black bag being less than under normal bags .

suggest that the exclusion of light affected fertilization^ Table 6

presents data, showing t emperature to be reasonably constat under both ,

types of bags so,that .presence or absence of light was the immediate

environmental factor evaluated and further suggests the necessity of

a light source © Table ? is a Comparison wei^it of seed produced,

under black bags and normal pollination bags©

Although these results are in opposition t© those reported by

J^ryangar and Roe (1936)s #io reported heads, dusted with sorghum pollen

in total darkness for '2k hours resulted: in complete sterility^ these .

results are in partial agreement with Stephens and QUinby (193k) who TABIE 60 Gomparisoa of . Tezaperatwe' (0eo) Eeeerded Inside Somal Bags: , and Black Bags.Used t© Gover Sorghw Heads0 Tuesonp irisom »; September^ IfSlo. „

* .r _r ^ • - r - i - O ' - ' v ’ -*0 0 * » " ^ O •• ' * " O -- • " ^ 4) 0 ' » -• 1* > O 0 -■ * •* •* *• lomalsBlaekfBeiiatiom: iSesraad. sBlaek sSevia*l©n$|H,ormsii sBlaekaSe-riation

. Bag l-Eag i\ ' §s ,Bag ai Bag s . ' ,? ?..Bag k Bag fL

s . . s ' ss> . ' 8 . ; 8 8 8 . 8 . S „ , . , • .-S .,, .. } .. 1 r’ ; ,s . , . . . . S . „ , , , : ... 88 ...... ■! . . . . . 1 286® j' 28©5«; 3-8?,2§&$ s: 27o5i s*X©.0 88 220® t. 20608 m2e# S :s s. 8 8 8 ' - ! 1 31*5 i 29»@| 2%$ ! 2io©i 00 .88 22o5 21o5|: 8 : ss t 8 . 88 S J 8 33*5. ! 31008 ^2 <50 ; 3 g 39*5 i .29*08 :~lo5 88 25oO 5 23 0 08 i 8 '’; - \ '' ...i! ! ' 8 8 8 . s -8 3ke® 32*0? =2o0' !: 32*0. s 30*58 -1*5 8 8. 25*5 g:2|j.o08 ^1<,0 s : s ' ' is s ! ; .88 ! 8. ''3t*fi5-*32*25-?' '- SI 3l»o® f =t0® ; st 26.0 i: 2ko5 f: : i !! 'f i 8 8 • | 8 3ho5 !' 3k*$ i 32o0i - ^2o0 8 8: 26o5 ! 25=08 ^Io0 . •i \ ■ v 2 ! s 8 8 i i 35»5 ? 3 W s *»3-oO - . si 35 #5 ? 3Wi: ?K .27*5 T 26=08. ^le0 i l ; ■ ■. ., S3 ? s ; ■■ ■ 8 8 f‘ ..? 36 b® 36001, % 3 ^ # ::: 37*5 ^ ?. -100 8 8: 28 60 2.7*Oe ^le© % , , | s s s ! 88 ? 8 ■t / s ss s s . 8 8: 29*5 sr28 0© 8.. =1<&0 ^ § ss s -S : : 1 8 ' ‘ '■‘■1 ! ! I!! 8$ 31*5 t 29o0f ^2 00 - i !? s 8. ?! ■ ! t '■ I ? ' s s s ' s 88 32O0 s 30*08 ^2o§ i . § _,ii ■8 : , i 8 8 :! .8 ; ,?"■ ' ? ss S ■ " s is 330© s 31*08 s S ■i / ' 88 ■ ■s 8 ! s ss S : • s 88' 3ij.e5 s 32*08 «2.©0 . • s : . s s s s 88 ., s . . 8

.Average ' . ' =Xo$6®Ce'' ' , ' *>1^3 ■" ■ " ': *1»731.

1 e l 0577eG0 Ii3

TABLE 7e Comparison of Seed Weight of Sorghm Seed Produced Under Normal P@lliBa|ioB Bags and Black Bags01 Tucsons Arigona <» ' September 5 1961© , / : 7 ..7 _ ‘

-• - • o- » - ' ... ■. . 7 . . , . . 1 - ■ ..-'■s/.. Seed Weight :In Grams s. . Variety , • ! - • '..v, ■- - - - ® - , § Seed Weight ■ . . ■ - . ; §• r" :

w ' m ■ ' :■ 1 : ' I B # 7' : t Normal ■ 1 Blaele Bags i . , : ... -- - A .7 ' •<.; "" ' ' - ' - 7 ' Airiak K=10 ' '■ '' s 8o8 s: : W ; z £ U t ' ..■ :• • ; ' ! ' !■: t : ledLaa • XQo0 7 ■ ; S ■. 1 ^ @ t i5®oQ . g: f 8 : ■ legari - i- 16.% 0 ■ i z. 1 W * 6 s * i f m 6 m , 16%5 ' ■ 23 &B- g- Bl08 ; ■ 7 1 BS38 = ■ ? 9 m , s • 1U0O z 155®6 ' § i 7 7078 yl7#0^ 'I r.-ii;®;--- 6 L 7 - 7 ■ g : ■ ? Martin' , . : ■7 , kio? . ; • 1 t - " 'if:' ' .f ' '' i W " \ I . 62®5 ' : ' , : . ■' kk . .

investigated the effeet of light ©ii pollen germinating on stigmatie ■

tissue® Stephens and. Quinfoy. reported, only. 31a2 per cent seed set

on heads dusted irith pollen a t -2 sOO Se Ho$ and more than 50 percent

seed set was obtained when pollen was dusted on heads bet^reen $$00

£# M@. and 8 s:00fA* Mo They concluded that darkness had m inhibiting

effect on pollen geminati©na ' .

: Pollen Exposed to Air . -

Pollen exposed to air for only two minutes loses so much

moisture that it sbrinksj however^ shrunken-grains are not necessarily

an indication of none»riable pollen* Studies have proven that such

shrunken grains have the capacity t© germinate provided its moisture

content does not reach a critically low level® The relationship of

per cent pollen germination to per cent of total pollen grains that

shrink upon exposure to air is shown in Figure 11#

Pollen grains exposed, to airfor two minutes showed an-increase

in total germination compared to pollen exposed to air for one minute®

When pollen coated slides were exposed to the air more than two

minutes and subjected to optimum conditions for pollen germinations a

decrease in germination occurSe iftien pollen coated slides were exposed

to the air more than five minutes^ the grains failed to germinate®

The work indicated that there is' a critical balance between moisture

and temperature within the grains if germination is to occur# + +

100 ‘ •• • Pollen Germination

90

80 *

70' tj

mi 60' ■s c $0" s a* h0'

lo­

go

10

FIGURE 11, Relationship of per cent pollen germination to per cent of total pollen grains that shrink upon exposure to air, Tucson, Arizona - August, 1961. BISGUSSIOH

ProbaMy the most' obvioms e onqlmsioa to. be derived from the

various studies sith Sorghum vulgar© pollea is the extreme delioaey

of the moisture relationship of the pollen grains and their

gerrainatioa®. • The sails of pollen grains exposed to air for more than

■ . ' two minutes apparently collapse through loss of water| whereas?

grains exposed to a saturated atmosphere itohihe water' ;s© f ast that

they hurst in less time® These same changes have been observed by ■

Sarioris (3^k2) in his work with Zea mays, v h m he , found pollen left

; under natural environmental eonditions .loses from W to $0 per cent

of the moisture in a very short period of times,

' : Anthony and larlan (1920) - report that barley pollen may lose '

a higher pereentage of water . than corn in the same length of time®

Observations of this study indicate that sorghum pollen is at least

as delicate concerning loss of water as-barley .pollen® '

The micre^elimate .of sorghum, pollen grains probably has a

significant bearing ©n pollen germination and subsequent pollen tube

growth o For purposes of this discussion^ the miero^climate ; refers to

that climatic area around pollen grains3 tiiereas^ the Hiaero=elimate

refers to that climatic area around the sorghum plant or in the fieldo

FflAen studies indicate .that.

grains are being transferred from the anthers to the stigmas the

zffiero=elimate around the •grains change0 Presmaablys the pollen grains

undergo some sort of physiological adjustment: in order for pollen ' gemination to ©ecmr0 Studies indleate .this adjustment increases the

per eeat; ©f peilea ^ genaiaation© - If this physiological adjustment in

the pollen grains does met ©eeurg pellem germinatiem is hindered to

seme extents ,

Sorghum heads failed to set seeds in the greenhouse when the

relative humidity was maintained above 80 per @ent<, tinder field eon^

- ditionSs anther sa@s failed to dehisce when the relative humidity in

the maero^elimate approaches 100 per eento Ihem■the relative, humidity

in the Biaero«eliinate approaches 90 to 100 per eents there is not an

apparent differential 'in humidity in the mier©=elimate of the pollen

. grains@ As observed under suoh eonditimsy pollen grains apparently

burst prior to or during anthesiso If dehiseene® of pollen grains

from the anther saes eeeur and the relative humidity in the. macro®

climate is relatively highs usually above 85 per eents the gradient ’

in humidity from the maerowelimate to the mdero^elimate renders pollen

grains B©m®viables because pollen grains apparently make corrections

in their own changing micro«elimate» There is a possibility that

pollen grains absorb moisture and bursts or absorb moisture and be®

come ,!heavy?i:s thereby loosing their ability to. flow freely«

;■ . Hypothetically speaking^ if the relative humidity at the, time

:, of.anthesis in a, sorghum’field is approscimately 70 per cent and the

sorghum field is irrigateds there is a possibility that the humidity

in the mer6=elimate will approach 80 per cent or higher® Consequently

the pollen grains would burst prior t©9 or during dehiscence and

pollination would fail to occur# - '

\ .Heseareh; results from the Arisons Agricultural Ezperimemt . . 2i8

StatioB show that in the luma Valleys liana j, Ari2onas one ©£ the major

■ pFOblema im the pr-odnetioa ©f grain sorghum in- the summer months is '

the failure to obtain sufficient seed seto Usuallyg the environmental

conditions that exist in luma Valley in the summer months ares (l)

. 'temperature at the time of anttiesis is relatively highg (f) little or

• mo m n d is present,, and (3) low relative humidityo Pollen apparently

• loses so much moisture tjixen anther saes dehisce that the change in

moisture from the miero-=>eliraate to the maero=>@limate renders the pollen

grains Bon^viableo

On a hot dry morning^ when anthesis is @@curringg the surface

cells:: of the. stigma tie tissue is .probably devoid of moisture^ there®

f©res the stigma cannot compensate for the loss of moisture from the

pollen grains» A proposed study in the sorghum^growing area of luma

Valley would be to irrigate a sorghum field coinciding with anthesis

in the-hybrid-or variety .planted® Irrigation should increase the

humidity in the micre»@limate *idh would increase the potential for

pollination to occurs A part of this study is to develop a technique

to determine viability of pollen grainso According to available

• literature2 the stmining technique for measuring the viability of; : ■

pollen is accepted by certain physiologists0 this technique is based

on the assuB^tioW that stained pollen grains measures starch content

and that starch content is correlated with the.viability of pollen®

Tests were conducted using the staining technique to determine the

percentage of viable and 'non^viahle poXXeno .:.S .random counting method

was employed in counting stained and unstained poUen grains 6 Ihen

a drop of iodine or aeetoearmine is added to a pollen«*coated slide^ the-"pollen grains tend to to the edge of the drop! eo&seqnent”

i y 5 httndrede of pollen grains congregate in one plaee5. making random

cotmting rather 'diffienlti

tFpon the completion of the stainahility trials =, other more

aeeeptahie methods were developed so that the viability of pollen

could tie determineda The most r ©liable, methods to dates which can be

used to test the viability of pollen9 is to check gemination percent-

" age of pollen grains in vitro' and in v It o q Grains germinated in vitro

.. means that nutrients are supplied exteraallyc Grains germinated in

vivo means that pollen grains supply their own nutrients- and depend on

environmental factors to initiate germinationo Fallacies have been

reported in all methods of checking viability of pollen except the '

test for in vitro and in vivo germination^

v' ■■'■■.■'Jalari and Vasil (1961) reviewed articles by .Androneseu^ and

■ Kearney and Harrison (1932) y im whieh they described the bursting of

pollen in a nutrient solution as ^pseudo<=>germination and ejection1^

Since . sorghum pollen has a high affinity, for water and will burst

easilyg the method postulated by Kearney and Harrison (1932) appears

to' be an acceptable method' for the evaluation, of viabilitye This ■

study indicates that this is not true for all pollens^ for example^ some

■; pollen grains in the Cueurbitaeeae family do not burst when placed in

water .0 In such a eases the staining technique would appear to be ' .1 •'" ■- ' ' : ' ' • ' - ■ ■ . . v " ■' -: :: - ' : ’ appropriate® , .

Sorghum pollen germination studies indicate thats at the mosis

35 per cent of the pollen grains were artificially germinated using

the in vivo method0 Attempts to germinate grains to vitro failed® From germinatiem studies aiteapted# sorghum polleu grains apparently

do not reqpire an external source of nutrients for germination or for

pollm tube growthp». Pollen grains in the Gramineae. family that have

been suecessfuTLy germinated in vitro on agar plates with different

poneentrations of sugar supplied ares C o m (Bailr, 19itl)» rye and

xdieat (Firbashs 1921)S ' and sugar eaae- (Venkatramang 1922)e 'Barley :

pollen (inthony and Harlan, 1926)' is ah exeeption in that it will, not

germinate using the in vitro method but will sueeesffully germinate

in vlvoe - ; \ : / - . ^ . ,

When amthesis ooours in a sor^ium head, millions of pollen

grains are liberated® The average nu#er of stainable pollen grains

eolleeted for any hour d.urimg this study was approximately 95 per :

cent® If stainable pollen grains in this study were a valid test for

• viability, then 95 per cent pollen geraiination should be espectedo

Sgains the stainable technique must be c onsidered invalid as a test

for sorghum pollen viability because at most, only 35 per cent pollen

-was germinated, artificially* This particular study indicates that

starch is present in 95 per cent ofthe grains0 Johri and fasil (1961}

reported that Bajpai and Lai .considered pollens of several drop plants

to be viable as long as they stained with aeetoearmine0 Unfortunately,,

this assumption is not always dependable, because as Vasil (1958) and

King (i960) report that fresh as well as preserved pollen, irrespect”

lye of whether they are viable or non=viables often stain with aceto»

on the viability of pollen grains, but on the content of pollen grains© fh@ highest per ’.©eat', of. mstained pollen grains oeetirred tshen the iaerease ©£ meraiag temperatufe amd the deerease of hmnidity - reaches seme'given'point developing a favorable environment« This.is an imdieatioa of the extremely intricate balamee between the environ* mental and physiological faeters of sorghwi pollen grains®

■ la this study when ■ germination eorats were made in ten minutes the TOgerainated grains if ere. resubjected t© ©ptimm .ar-tifioial com™

ditions necessary for germination^, When the grains were again re=>

examined very few of the remaining grains germinatedo Thus it appears

that ihen humidity^ t©mperature9 age of pollens and other physiological processes all arrive at some prescribed levels a ^trigger mechanism^

operates and a percentage of these pollen' grains capable of germinating

do so a This could possibly account for the small per cent of total pollen grains that germinates Pollen grains germinated best Wien the

humidity in the chambers and in the petri dish was 92 per cent relative humidity at a temperature ,of 85e F® These studies have proven that the physiological responses of pollen grains to relative humidity are 'ex*

tremely critical^ Anthony and. Harlan (1920) observed a tiny drop of water on the stigma.tie tissue of barleyi whereass close observation of

the sorghum stigmas failed to show this* Just prior to and during

anthesisp sorghum heads appear to contain more moisture than at any

other time® More elaborate studies are still needed to determine & , more exact relative humidity and temperature range for optimum pollen

germiaationv ' '' ■ ' ' .

, • '. pollen grains exposed to air for more than two minutes lose s© much moisture that they become shrunkeno- Repeated studies show,.. how» evers that a eertaim amoumt of dryness is necessaiy to obtain optiimm

•gerainati0n$ and thds ooenrs when pollen coated slides are exposed-to

the air for,tie imrat@s0 TShen these same shrunken grains were placed

'id a saturated atmospheres they absorbed moisture and attained the

round appearance of normal pollen grains but again failed to germinate*

The normal physiological make-up of the pollen grain compensates for

miner changes in environmental factors^ but once a critically low level

: ' . - " ■' .... ' : ■■ - ■■ - : . , ■ : . ■ ' . is reached^, the pollen0stability to germinate is destroyedG

Pollen failed to germinate inAa control chamber at 92 per cent

relative humidity and 8j> . Fo. when light, was absent# iliem fluorescent :

light was presentg no germination occurred until additional light was

supplied from a T5 •Watt .bulbo' This led to the conclusion that a certain

wave length of light was requiredb A mere elaborate study is needed to

determine conclusionss because there is much conflicting, evidence re­

ported by various scientists in this particular field of study» . Re­

sults from the study concerning.the effect of light on pollen

germination in the field on- stigmas'are not in agreement, mt h the re- •

suits obtained in the laboratory, in whidi partial germination occurred

in the presence of light3 but no germination occurred without light0

The weight of seed produced ©n varieties and hybrids of sorghum in

* i c h light was excluded varied considerably<> Inconclusive results,

howevers indicate that light has some inhibiting effect on pollen

germination of some;, varieties and no. effect on others 0 Ayyangar and ■

Roe (1936) reported complete sterility when pollen was placed on sorghum

heads in complete darkness| whereas3 Stephens and Quinfey (193h) reported

''SB" per cent seed , set when heads were dusted with pollen-at 2 sOO J.e Me jlH combined information at present, leads one to believe that lack

of light has some inhibiting effect on pollen germination^, but to

* a t degree has not been determined® therefore$ more elaborate

experiments are necessary to determine if light is ah essential

physiological factor for pollen germination®

The aggregation of pollen grains bn a slide has a marked

effect on the germination and length of pollen tubes® Such pollen

grains send out short pollen tubes compared to tubes from grains that

are not crowded® This can be compared to the ,$b±sS effectof yeast

and otiier microorganism that the growth of the organism is stimulated

when the inoculum is heavy® According to Brink (192lj.s #2 and #3) the

stimulating effect on pollen germination is presumably due to certain

growth promoting substances which dif fuse out of the ■ pollen grains &

The in vitro method of pollen germination was not used

successfully in this: study®. Two suggestions are listed which should

add additional information concerning pollen germination on nutrient

agar solutionsk

(!) Place f reshly made agar plates or agar coated

, slides of the various solutions i n a pre-

heated chamber at 85 F® with the relative

humidity set at 92 per eent® ■

(2): Germinate pollen grains in the natural environ^ /

meat of the field® -

One of the unforeseen problems in this study was in the storage of the

nutrient agar plates and slides®- Agar coated slides and agar coated

plates were prepared but not stored .in preheated control chambers, at a ' definite; humidity before usec Therefore^ they lost moisture as they

were allowed to cool to temperature ranging from 70° to 8&° Fo The

possibility exists that the sudden physiological shock to the pollen

grains may hare r endered them non-riable because when placed on agar

coated slides or agar plates9 they were inadequately supplied with

moisture;or too;cools; ' : h \

,,,; On Pages 16 and 17 a method is discussed in which sor^ium

heads in the laboratory can be induced to supply pollen up to fire

days© Pbllen produced at arithesis the first day has a higher percent­

age of viable pollen than that produced on subsequent days© When

sorghum heads are cut and removed to the laboratory^ there appears to

tie a physiological adjustment between the pollen grains and the stigmas

in the top one=third of the head allows fertilization to occur© There«-

afterg fertilization is rare; indieating that sor^mm pollen loses its

viability ti eeause . t he turgidity of the pollen on the s tigma i s lost © ■

Size of pollen grains probably offers one of the most

interesting, fields of study© According to Hikkelson (19|i9f temperature

has little or no effect on pollen grain size© Studies Could be made ,

that could answer questions concerning relationship of pollen grain

size and genninatioh and whether difference in size is due; to the

environmental or genetic factors® SUMSEY AMD mMCLUSICBf

'. Studies, investigating the artificial germination of Sorghum vulgare pollen and the effect of environmental conditions ©a pollen germination were macteo Goimaerclal varieties and hybrids were used ' in the various studiess G.embiae Kafir 60 was selected for germination studies because of its adaptation to semi*»arid regions and its homozygosityo The plants were grown at the University of Arizona

■,Campbell.Avenue Farm2 TucsonArizona* Laboratory faciHties, irere - v located in the Botany laboratory and in the Plant Breeding green=> housee This work is of a subjective nature and observations and ©on-=

.#us$ons.are-listed* ^ - .o.'-.

■Conclusions •* ^ > •' li,. Sorghum heads can be removed to th.ep.aboratory '1 - . and handled in such a way that anthesis' mi l

©eeur continually for five daysQ

2 a Hhen anthesis occurs on cut sorghum heads in

the- head house s pollen grain viability is good; ■

the first day but each d^r thereafter decreases*

3® Sorghum pollen germinates in vivo o .

Uo Exogenous nutrients- are not necessary for

sorghum pollen germination or pollen tube

growth®• '' : ' ' /

5o .Humidity is the most critical environmental

factor for pollen germination* . 60 Pollen grains geminated best when plaeed ' ,

in an artifieial environment of 85 F® and

92 per cent relative humidityo

To' ^ Sorghum pollen r equires a period of dryness ;

for optimum geraiinatiomo . .

80 Iodine stainabilitys as a test ©f viability

of sorghum pollens is questionable® There

may be no positive relationship between the '

number of pollen grains that will germinate.

and the number that stain with iodine#

Observations • : •- ■ /;. ; • " v. '' ... - V

lo ; Pollen grains 'placed' ’in 15-20. per cent sugar

(sucrose) solution formed small knobs by the

protrusion of ;the cell content from the pore /

through i-Aiidh germination■ takes place® '

20 The daily pollination period of Combine Kafir 60

■ variety occurred between 6 sOO % 0 Mo and lOiOQ

Ao -H© Slight variations of this period were

caused by fluctuating environmental conditions e

3» Approximately 9$ per cent of the pollen grains

collected from 6 s 0® A© M». to 10x00 A© fE© stained '

with iodine# : " ■ /' ; \

It© Pollen grains have an extremely high affinity for

moisture which is rapidly lost under dry conditions0

;5i' the relative humidify in the macro-climate is

above 90 per cent pollen grains fail to freely deblsee from the anthers sae0

6t0 Sorghm heads appear to be more moist during

m d prior to anthesis than at amy other time©

7@ Sorghmn pollen grains generally germinate

within ten minuteSo , : •

8© The germimatiott of the pollen grain and develops

ment of the pollen tube was fully deseribed with

mierophotographs and explanation of the phenomena

observed© LITEMTUEE CITED

Mdieott, Fo T6S Pilleii gmiiiiaaildn and' pollen' tube growth as ihflueneed"by pirre growth substanceso PQ Physiology . x ;_;;18s270-279.».' , v" : . V;.:.'; . ;......

Anthonyg S os and He 'Go Harlan^ 1920o "Barley Germination» Jour0 Agrico ' - .Bes? Vol»18d25^3S» -

.Srtsehwager9 Is- 'H0j> l<%8o Matony' and ‘morphology 'of vegetatite organs - - ■ -: - of -Sog^ma' 'valgare"plant indmstryc. Soils and Agrio Enggs Bureau liais e s © -

Artsehwager j, E e Ros ahd"Ra" 'Go 'McGuires 19k9o Cytology'of Reproduction ih'SorghtM TUlgaree • Jour0 : Agriee BeSo Tol® 78s :" ; . 669^ 73, . ;•

Ayyangari , Gs Ho R e, and V. Pc Kao,1236. Studies in Sorgh-um - The ' Indian Great Millets" 'Ili ihtKerSj, 'pollen and stigmao : v . ■ Indian Jour0 Agr. Seio ,6sl299-=13220 _ ' /

Bair3, Ro Ao-s and We Eo loomiSj, I9I1.I0 , The. germination of maize pollenc. Science :1684169a

Bennett^ Ho Wo9 1959o ;irtificial'pollen germination for selection'of' improved" seed prdductidh in Paspalum dilatatum, Poir0 Agron0 Jour0 5lsl09=-lllo

Brinkj, R o9 192)4 o The physiology" of 'polleno- The requirement for growth^ #le Amero Jouro Bots lls;2l8=>228#

' 4 192)40, The physiology "of pollen0 Fur titer • considerations re= gar ding the requirements for growth a #20 Amero Jour0 .. . . Boto 111283^0. . • . ... , •

. a 192)4® The physiology'of 'pdllerio' Growth in vitro and in vivo . - #3® . ..Arner,® Jour® B.ot# 115^51^36)4© - ;

' a 192%® 'The'physiology of' poHen0 ' Chemot'rdpisms effect on ' gremping "Of grains3 "fomatioh' and function of eaUose plugs| summary, and conclusion:j, #ii,0, .Mer ® Jour 0 B©t@ ' . . 11:1417=43^ - / , . .:

Dandliker^ Walter B®9 ¥illiam Co Cooper^ 'and Hamilton P® TraUBs- 193 % ., . Vitamin & and the germination of pollen® Science ;; .■ ' . ; 88:622® ^ " '; . 'Butcher j, R® Acs 1918» " fxtamtn, Siiidxes' HI ohservatidn oh the curative . ■ : properties of hohe,ys nectarj, and corn pbll'eh iu avian ' polyneuritiso Jouro Biole Gheme,

Brdtmaug Gxmnars 19h3*>; Ih latroduetioh To -Pollen,. Analysiso ¥althams ' . Hasso Chronica Botaniea. Go0 .

■ . ; a. 1952 o . Pollen' Morphology ahd Plant Taxonomyo ¥althama ,: ; Masse ■ Chronica Botaniea Coo

Firbashs Heinrich^"1921o' Bye and'wheat polleh germihatioho Zeitschri . ■ . ; Pfanzensudht S ;70=73o Botanical Abstracts Volei 9 s1325s 1918

Flozys Walter$ ' and M0 Bo Tomas, 19%3, "Studies' of plum pollens. its; " " .. _ : 'appearance and geminationo Jbier0

Golubinskia I0 Wos 19$0e The effect of the corella oh the gemmation ': - o f poilm, grains (Bussiahlo Sad i Ggorod (Orchard - #d\GarW) #A#^lo'' ' f ' ' . ; -

Got

Handbook of Chemistry and' PhysiCsa l|.2nd."Editions I960® Gonstant Huiniditya p® - 2J4,99“2900o

Hellmersa H«,s and ffichlisa 'l936e, ' Exogenous substrate" utilization and geminatibn by the pollen of Finns ponderosa® ■; /V/'-, Plant Physiol^g 31:28^2#® : ' ; dohrig B® Hi, aud io K0: ¥asxla . 196lo 'Physlblogy of pblleht ;¥ol-e '27s #3> Bniversity of Hew Delhis July=Septembers 1961®

JonesSr Helvin Bes atid do Co Bf oims; 195lo' Pollihation cycles of some ... grasses in Oklahoma» Agroru Joura l0s;2l8»222o

Harms/Fo Jes 19l5o Germinating polleno Science h2 ;6l0~6llo

Kearneya T e Hoa. and'G0 To. 'Harrisona '1932o Pollen antagonism in cottonQ Jouro Agr® ;Beso:? 1^:191-226« - . '

Kingd Jo Roa I960e ' The 'pefoxidase 'reaction as' an. indicator of pollen " •- viabilityo Stain Teche 3$:22$«227o

Enomtbng. Ho; E=s 1922o' Studies in pollen witii speciaT reference td" ' longevity® Cornell Agre Expc 5ta0 Hemos 52i?5la793o. Martin9 John Hcs 1939o'' "Sorghran Improveraeat-Iear Book of Agricultures

•: ■ ■■:.■; ■•• . : ■ s Glimate' # 6 " (scrghw)% Year. Book of ; : Agrieuatnre,9 p<;3U3^7»;'-;._,vn/V • j

■ . ' : . ', ' Crops' 'ia Peaee'and War ’.(sorghum)® • Year Book - I,:.. ... -... rf AgriaiIWei,_i,„:173,. ■ - ' '

Martin^ John Hoa C®' Stephens^ 1955® 'Culture amd use of sofgum for forage® Barm® Bui®.lBWtgP@k2 (reYa supereedes • farm bui#. H58)@ ■ ' ■ ...; ' . . ; 'r:" x-*;"V' \ ..' Martin^ J® M ss-1913»' The physiolo^r of pollen of Trifolium pratehseo ; ' :. , . Bot0 GaZos 56:113-126^ -

Martin^, Jo l=s and U Eo Yocums 19U0o I. study of the pollen;and i"' \ ■/■'■■|>istxla;^.i'applesViH’-re^‘^©nr.tbvthe^ermiHatic^..0Y . ■ ; pollenc Proce Iowa Jkea.0 Scios 25s3U0m391e .. ■■ ■'

Mikkelsons Valdemars"19h9^ "Has'temperature'' any influence op pollen .siief' l^grsiol:e.'Elant»s v2i323»32ii,o

0*Kelley^ Joseph Cos 1955e :Epfcerrial: earboKydrated; in 'growth/and/ ' /. ' ■ respiration of pollm tubes in vitro o Amero Jour® : BotQS h2s328*=327» ' :% „ : , - ■ - ■ - -- '. , ■. • 1 .' ■" ; ' , ;; ' . . ' : . J 1957® Boron effect on gro>rths :bipygen uptake ind sugar "absorption by germinatione Jteer*. Jdar ® Bote toK239-2iih® ; .

Pooleg Charles Fog"1932® Pollen grain studies"as an indication of fertility in hybrids® Genetiess 17:125^236®

Popes Merritt. E#s 1937# Ihe"tihe f aotor" in pollen tube growth" and fertilization in barley^ Jour® Agrie® Pes@ dk: . 525-529® ■ " V

Raghavans. Ves and':Hs' K® Barushs 1959/ Effeet oftime factor'on the ■ stimulation of -pollen germihatlon'^and pollen"tube ' ' growth by eertaih auxins3 vitaminss and trace elements . Physiologia Plamtarums 12i:#l.»Ml/

Sartoris $ George B// 19h2@ longevity of.sugar cane" and corn poll eh®. ' A . methdd'f or"long* distance' shipment of sugar cane pollen . ' by . airplane e Jmer e Jour o' Bot® 29 s395“h0Q« > / Smiths % s and H 0 'lo' Cbchrahy 1935®//Effects of temperature on'/. germination and tube:growth in the tomato®" Minn® Cornen Agric® l2^/ S*aos 175^ lis Bible'7o . " Sxnit-hg B o , E o 9 1 9 3 9 o "'The"infliieHee'@ f 3«i5.dola :acetle acid on pollen ■ V, ;. grain; germination.». Seiences 90»l63al61u

Spencer s Mo H 0# 19260:"" EaBoraiorymetilibcl" f o'r'mintaixiing'" const ant'- ' , . humidity» Internat.ional Gritieal Tabless Vol0 1..$ F o:.67=.68o '

Stephensg and Jo RG Quinbys'l93lie- 'Anthesiss pollination^ and . fertilisation in 8orghum@ Jour0 Agrico Res0 k9° 123=lk6e • ' ' . . , ■ ::<

Vasily lo 1958= The e tE tiiiation of excised anthers and the ' . culture and. storage of pollen: grains» Ph= Bo Thesis^ ;u' ; 1 Eeihi: UniTe India# ^ . - ■ . -, - , ,

¥enkatramans B 0 A‘=s 'and Sahib To So Eads 1922; Germnation a M pre~' serration of sugar cane pollen® Agric® Jour0 Bidias .

:Vi#la A® J o Co;Stephenss 'aha::«Jo'Ho'Martin^ 1936o '.Tdentifieatloh' ■" -h ■ ; history'ahd -distribution of eomraon i'Orghum varieties* • USD! Technical Bulletin Ko0 5o6s 1=100

•:... Visser2 To s 1951* 'Gerraihatiori' and"stora'g’e of pollen = Meded Landb - ' . Hooquseh (Wageningen)@ 55sl“68a

Ward. Jesse 1*^ 'andrEeVi;: Srewder^ :'l9Sli"r 'The' 'graLii sorghum challenge ' • / Jr^ared‘''l^:'T(^¥!Elecfe&e"Serviee operation with the 'Agriculture Committee 2 West Texas ■ ''.V;-' Chamber of Commer@6® \yi, : ,

Wodehouse2 Roger Pb9 1 9 5 9 @ Pollen Qraihsg"Their Structure2 Identification 1, ,Wd-Sigmifieamee. in Sciemce and Medicine0 Rew York - ‘ ■ Hafner Pubo Co* «\v , •_ ' \ ' ' { \ t':'': :':

- 4,

"i O' .