In vitro morphogenesis in peanut

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Title:
In vitro morphogenesis in peanut
Physical Description:
viii, 79 leaves : ill. ; 28 cm.
Language:
English
Creator:
McKently, Alexandra H
Publication Date:

Subjects

Subjects / Keywords:
Peanuts -- Morphogenesis   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 74-78).
Statement of Responsibility:
by Alexandra H. McKently.
General Note:
Typescript.
General Note:
Vita.

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Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001477681
oclc - 20957567
notis - AGY9605
System ID:
AA00002147:00001

Full Text











IN VITRO


MORPHOGENESIS


IN PEANUT


ALEXANDRA


MCKENTLY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
















ACKNOWLEDGMENTS


I gratefully


acknowledge


the


support


of Dr


. F.P


Gardner


during


course


of study


Thanks


are


extended


. G.A


conducting


. Moore


thi


her


ass


project.


instance,


Thanks


advice,


are


also


and


insight


extended


to the


other


members


my committee,


. K.J


. Boote,


. P.S.


Chourey,


knowledge


. D.A.


and


Knauft,


assistance


and


they


. T.W


provided.


. Lucansky


the


acknowledge


the


financial


ass


instance


provided


the


Walt


Disney


World


am especially


grateful


the


confidence


and


support


that


received


from


husband,


Barry,


while


attaining


thi


academic


achievement.
















TABLE OF CONTENTS


pace


ACKNOWLEDGMENTS............ ....................... ii

LIST OF TABLES ..................................... iv

LIST OF FIGURES.......... ........... ............... v

ABSTRACT....... ............................. ........ vii

CHAPTERS

1 INTRODUCTION.................................. 1


2 IN VITRO PLANT REGENERATION OF PEANUT FROM
SEED EXPLANTS....................... ..........

Materials and Methods.........................
Results and Discussion.......................

3 ORGANOGENESIS FROM CULTURED LEAF TISSUE OF
PEANUT AND PERENNIAL PEANUT...................

Materials and Methods.........................
Results and Discussion........................

4 IN VITRO CULTURE OF PEANUT ANTHERS..........

Materials and Methods.........................
Results and Discussion........................

5 SOMATIC EMBRYOGENESIS FROM IMMATURE
COTYLEDON TISSUE OF PEANUT....................

Materials and Methods.........................
Results and Discussion........................

6 SUMMARY AND CONCLUSIONS.......................

REFERENCES.. ......................................
















LIST


OF TABLES


Table


Page


2-1.


Peanut genotypes used
cultures.............


for
e.t..


seed explant


2-2.


In vitro production of
shoots, as influenced
(BA) level and explant


Florigiant peanut
by 6-benzylaminopurine
type...... ............


2-3.


2-4.


Shoot
ledon
levels


production from whole
explants of Florigiant
of 6-benzylaminopurin


Comparison of mean
whole embryonated
giant peanut on so
plemented with 25
L~- over time ....


embryonated coty-
peanut on varying
e (BA)............


shoot production from
cotyledon explants of
lid and liquid media,
mg 6-benzylaminopurine


36
Flori-
sup-
(BA)


2-5.


Morphogenic responses
culture..............


of peanut


cultivars
..... .....


in
....


3-1.


Peanut


genotypes


used


leaflet


cultures...


3-2.



3-3.


Morphogenic
explants to
aminopurine

Morphogenic
cultivars in


response or
varying con
(BA)........

response of
culture...


Florigiant
centrations


leaf


explants
.O.*.....


peanut leaf
of 6-benzyl-
.............


of peanut


3-4.


Morphogenic response of leaf explants from
perennial peanut cultivars Arbrook and
Florigraze to varying concentrations of
6-benzylaminopurine (BA).......................


4-1.


Relationship between
microsporogenesis of


flower bud length and
three peanut cultivars....


4-2.


Mi crnmannorp


ullrvival and


devsl nnmsnt


nf
















LIST


OF FIGURES


Figure


Page


2-1.


2-2.


2-3.


2-4.


Multiple
surface
explant


Plants
whole
in vit
25, 30
purine


shoot production from the adaxial
of a whole embryonated cotyledon
of Florigiant peanut...................


of Florigiant peanut regenerated from
embryonated cotyledon explants cultured
ro on media supplemented with 0, 10, 20,
, 40, 50, and 60 mg 6-benzylamino-


Multiple shoot production
onated cotyledon explant
cultured on solid medium
mg 6-benzylaminopurine L-1


from a whole embry-
of Florigiant peanut
supplemented with 25


Multiple shoot production from a whole
onated cotyledon explant of Florigiant
cultured in liquid medium supplemented
mg 6-benzylaminopurine L- .............


embry-
peanut
with 25
........


Bud tissu
face of a
cultured
1-napthal
aminopuri


originating
eaf explant
medium supp
eacetic acid
SL- ........


from the adaxial sur-
of Florigiant peanut
lemented with 1 mg
UL' and 3 mg 6-benzyl-


3-2.



3-3.


Shoot
giant
with 5


differentiation from bud tissue of Flor-
peanut cultured on medium supplemented
mg 6-benzylaminopurine L1 .............


Organogenic leaf callus of
peanut cultured on medium
mg 1-napthaleneacetic acid
zylaminopurine L ........


Arbrook perennial
supplemented with 1
L-1 and 3 mg 6-ben-


3-4.


Shoot differentiation from leaf callus of
Arbrook perennial peanut cultured on medium
supplemented with 5 mc 6-benzvlamino-


3-1.









Fiaure


4-1.


Uninucleate microspores of Florigiant peanut
stained with acetocarmine after 7 days of
culture...................... ....... ..........


4-2.


4-3.


4-4.


4-5.


Densely stained binucleate
Florigiant peanut after 28

Lightly stained binucleate
Florigiant peanut after 28


microspore of
days of culture....

microspore of
days of culture....


Callus tissue originating within an anther of
Florigiant peanut and protruding through the
wall after 14 days of culture.................


Shoot structure arising from callus initiated
within an anther culture of Florigiant
peanut.................................... .........


4-6.


Shoot
within


structure
an anther


arising
culture


from callus initiated
of Chico peanut......


Page





















Abstract


of Dissertation


Presented


the


Graduate


School


of the


University


Requirements


of Florida


the


Degree


Partial I
of Doctor


fulfillment


the


of Philosophy


IN VITRO


MORPHOGENESIS


IN PEANUT


Alexandra


December,


McKently

1988


Chairman: F
Cochairman:


Major


'rank


Gloria


Department:


. Gardner


Moore


Agronomy


Peanut,


Arachi


hvpocaea


improvement


conven-


tional


breeding


methods


been


highly


successful,


although


current


limitations


progress


are


the


impetus


to alterna-


tive


genetic


research


was


manipulation


to investigate


techniques.


vitro


The


purpose


morphogenesis


pea-


nut.


The


areas


of study,


organogenesis


from


seed


and


leaf


tissue, a

chosen be

programs,


Lnther

cause


culture,


their


namely,


and


somatic


potential


the


development


embryogenesis


benefiting


, were

improvement


an efficient


plant


regeneration


system,


development


of haploid


plants


wide


hybridizations


with


normally


incompatible,


diploid


species


that


carry


desirable


traits,


and


the


rapid


production


of homo


zygous


plants.


- S S


I


-L


_


I









deembryonated


cotyledons


that


were


whole


and


sectioned.


Multiple


shoots


arose


on 6-benzylaminopurine


(BA)


supple-


mented


media


(0.5-60


mg L-) ,


with


maximum


production


occur-


ring


at 25


Shoot


regeneration


occurred


from


the


adaxial


surface


of leaf


explants


of peanut


and


perennial


peanut,


Arachis


alabrata


Benth.,


cultured


on BA-supplemented


media


(1-10


mg L-1)


with


maximum


production


occurring


and


The


excised


shoots


of peanut


developed


roots


upon


transfer


to medium


supplemented


with


mg 1-napthalene-


acetic


acid


In vitro-produced


plantlets


transferred


soil


and


placed


a greenhouse


developed


successfully,


matured,


and


seed.


No phenotypic


variants


were


observed


among


any


the


plants


produced.


Thus,


these


two


systems


allow


vitro


production


of morphologically


normal


plants


high


frequencies.


The


vitro


survival


and


development


of uninucleate


microspores


over


time


and


the


regeneration


response


cultured


anthers


were


evaluated.


Mitosis


and


segmentation


occurred


the


microspores.


Structures


resembling


embryos


emerged


from


callus


originating


within


anthers.


Root


organogenesis


occurred


from


somatic


tissue-derived


callus,


however,


whole


plant


regeneration


did


not


occur.


Somatic


embryogenes is


from


cotyledons


immature


zygotic


embryos


was


investigated.


Embryogenesis


did


not


I


I i


I I


m





_ ___


_I















CHAPTER


INTRODUCTION


Arachis


hvoocaea


the


cultivated


peanut,


a crop


maj or


economic


importance


as a source


of protein


and


both


developed


and


developing


countries


(Purseglove,


1985).

tropical


The

and


species


subtropica


native t

1 zones,


:o Brazil

as well


and


grown


as warm


temperate


zones.


latitude


Commercial


(Cobley,


production


1985)


found


Asia


between


largest


N and


producer


peanut,


followed


Africa,


North


and


Central


America,


and


South

large


America.

t producer


Among

in t


individual


he


world,


countries


followed


China


India

. the


United


States,


Sudan,


and


Nigeria


(Bajaj,


1984).


A number


of objectives


exist


peanut


improvement


pro-


grams


High


and


stable


yields,


drought


resistance,


high


protein


and


content


, and


earliness


of harvest


are


characteristics


being


pursued


in various


programs


(Bajaj,


1984)


One


the


major


objectives,


however,


to improve


the


disease


and


insect


resi


stance


plants


through


the


pro-


duction


of genetically


resi


stant


cultivars


(Rugman


and


Cock-


ing,


1985).


Commercial


cultivars


are


susceptible


a range


of pests


and,


consequently


, crop


yields


are


reduced


(Garren









attention


to wild


Arachis


species


which


possess


these


res


instances,


among


other


favorable


characteristics


(Banks,


1976).


For


example,


Arachis


monticola


Krap


et Rig.,


Arachis


alabrata


Benth.,


and


Arachis


villosulicarna


Hoehne


are


resistant


to Cercosnora


while


Arachis


harenbeckii


Harms


and


Arachis


prostrata


Benth.


are


drought


resistant,


and


Arachi


dioaoi


Hoehne,


Arachis


marainata


Gard.,


and


Arachi


villosa


Benth.


possess


high


mineral,


protein,


and


con-


tents


(Bajaj ,


1984


, Stalker


and


Wynne,


1979).


The


genus


Arachis


has


both


diploid


(2n=20)


and


tetra-


ploid


(2n=40)


species.


The


cultivated


peanut


tetraploid


and


not


sexually


compatible


with


the


wild


diploid


spe-


cies.


Thi


incompatibility


provides


a significant


barrier


to the


wild


transfer


species


of known


the


genes


cultivated


favorable


peanut.


traits


Numerous


from


interspe-


cific

but g


crosses


generally


have

have


been

been


attempted to bridge

unsuccessful (Singh


these barriers,

et al., 1980,


Gregory


and


Gregory,


1979,


Stalker


and


Wynne,


1979,


Johansen


and


Smith,


1956).


The


limitations


of conventional


breeding


methods


among


Arachi


species


have


enhanced


the


potential


of alternative


techniques


of genetic


manipulation.


Plant


biotechnology


becoming


an important


tool


the


genetic


improvement


crops.


Recent


advances


in genetic


manipulation


techniques,


A p -- r


.1 -


1 r? L _


SD.,


a. *


.


a









could


be applied


to peanut


improvement


include


the


produc-


tion


of haploids,


the


recovery


of genetic


variability


through


somaclonal


variation,


and


the


vitro


selection


mutants


resistant


to salt,


drought,


herbicides,


disease,


insects


(Bajaj,


1984).


The

dependent


success

upon a


of all


n efficient


these


system


vitro t

of plant


techniques


regeneration.


Before


any


meaningful


work


can


be done


on the


incorporation


these


methods


into


improvement


programs,


essential


that

ous


basic

peanut


information


tissue


on the


explants


regeneration


be obtained


response


(Bajaj,


of vari


1984)


Limited


research


has


been


reported


on the


vitro


ture


of peanut


(Bajaj


et al.,


1984).


However,


the


litera-


ture


that


does


exist


Arachi


species


indicates


a poten-


tial


the


use


of tissue


culture


techniques


plant


regeneration


regeneration


Bajaj


peanut


from


crop


seedling


et al. (1981a)


and


improvement.


explants

Mroginsk


Complete


of peanut

i et al.


plantlet


was reported

(1981), and


from


seed


explants


Illingworth


(1974)


and


Atreya


(1984).


Bajaj


et al. (1980)


reported


microspore


embryo


callus


development


from


anther


cultures


of Arachis


qlabrata


Benth.,


and


Martin


and


Rabechault


(1976)


obtained


anther


tissue-derived


callus


from


peanut


cultures.


The


overall


obj ective


thi


study


was


to investigate


* _


,,, _


- -- -- -- r -


m,


A


I


L


.








from


the


cotyledons


of zygotic


embryos.


These


areas


were


chosen


because


their


potential


benefiting


peanut


improvement


programs.


The


benefits


are


the


development


haploid


plants


(n=20)


wide


hybridizations


with


normally


incompatible,


wild,


diploid


species


(2n=20)


that


carry


desirable


traits,


the


rapid


production


of homozygous


plants,


the


mass


propagation


of Fl hybrids


, haploids,


and


other


rare


or desirable


plants,


and


an increase


the


available


gene


pool


utili


zing


new


genetic


resources


induced


tissue


and


callus


cultures.


The


following


four


chapters


deal


with


research


plant


regeneration


via


organogenesis


from


seed


and


leaf


explants,


microspore


development


and


plant


regeneration


from


anther


culture,


dons


and


of zygotic


somatic


embryogene


embryos.


The


from


final


the


chapter


immature


summarizes


cotyle-


the


conclusions


the


experimental


findings


thi


project


and


emphasizes


areas


where


additional


research


may


prove


produc-


tive.















CHAPTER


IN VITRO


PLANT
FROM


REGENERATION


SEED


OF PEANUT


EXPLANTS


Peanut,


Arachi


hvDoaaea


is an important


crop


the


production


of oil


and


protein


from


the


seed


both


developed


developing


countries.


Clonal


propagation


favorable


would


lines


facilitate


through


the


breeding


and


vitro

crop i


culture


improvement


f explants

programs


(Bajaj,


1984).


An efficient


plant


regeneration


system


also


a prerequisite


genetic


transformation


studi


using


Acrobacterium


tumefaciens


(Smith


and


Townsend)


Conn


(Horsch


et al.,


1985)


Plant


a number


regeneration


of legumes,


from


seed explants


including


mung


has


bean,


been


Vivna


reported


radiata


(L.)


. Wilcz


and


pigeon


pea,


Caianus


cai an


Huth


(Mathews


Rao,


1984;


Mehta


and


Mohan


Ram,


1980) .


Several


reports


have


described


vitro


plant


regeneration


of peanut


from


various


seed parts.


Illingworth


(1968,


1974)


reported


plant


regeneration


from


deembryonated


cotyledon


sections


cultured


nitrogen


on basal


treatment


medium


either


of seeds.


The


following


liquid


or without


nitrogen


liquid


caused


cotyledons


to separate


into


fragments.


Bhatia


et al.


(1985)


-3 - -- A- ,.. -.


I------------------------I- -,


- -3


___~__


-^ _


- _-









water


at 0.05


to 0.5


Atreya


et al.


(1984)


regener-


ated


plants


from


both


excised


embryo


axes


and


embryonated


cotyledon


segments


cultured


on medium


supplemented


with


BA L~-


These


studi


provide


limited


quantitative


and


com-


parative


information


on organ


yields


per


explant


type,


suc-


cess


rates


of transplanted


vitro-produced


plantlets,


and


the


productivity


of various


genotypes.


This


chapter


describes


an in


vitro


regeneration


system


that


maximizes


the


number


of plants


attainable


from


a single


seed.


The


objectives


were


compare


the


regeneration


response


of five


seed


explant


types,


determine


the


most


productive


medium


multiple


shoot


induction,


induce


root


formation


and


subsequent


growth


vitro-produced


plantlets,

cytokinin


determine


concentrations


the


long-term


during


culture


effects

stages


of high

on mature


plant


development


and


function,


and


assess


the


response


of various


genotypes


to this


tissue


culture


system.


Materials


and


Methods


General


protocol.


Mature


peanut


seeds


were


soaked


tap


water


containing


0.5%


antimicrobial


P-chloro-M-Xylenol


Seed


coats


were


removed,


and


the


seed surface-


sterilized


separate


ethyl


immersions


alcohol


sodium


5 min


followed


hypochlorite


for


three


4 min


each


on a shaker


Seeds


were


rinsed


three


times


with


ster-


S- *) a *


r i q


*


1 il 1


1 1


*


q









sliced


once


longitudinally


to yield


sectional


embryonated


cotyledon


(SEC)


and


sectional


deembryonated


cotyledon


(SDC)


explants.

explant t


Embryo


"ype.


axes


Explants


(EA)

were


were


excised


placed


into


to yield


the


either


fifth


X 25


glass


test


tubes


containing


20 mL of medium


or 100


X 15


plastic


petri


plates


containing


25 mL of medium.


The


nutri


ent


medium


cons


isted


of Murashige


and


Skoog


maj or


minor


salts


(1962),


sucrose


mg glycine


, 0.5


nicotinic


acid


, 0.1


mg pyridoxine


, and


0.8%


Difco


agar.


Various


concentrations


1-napthaleneacetic


acid


of BA

(NAA)


(0.05

(0 -


- 60


mg L-1)


mg L-1)


and


were


added.


The


the


media


was


adjusted


to 5.8 with


potassium


hydroxide


or hydrochloric


acid


prior


to adding


the


agar


The


media


were


autoclaved


at 1.1


cm-2


20 min.


Cultures


were


maintained


at 23C


on a 16/8


h light/dark


cycle.


used


General


which


Electric


produced


F40CWeRS*WM


approximately


fluorescent


98 gmol


tubes

when


were

mea-


sured


mm from


the


tubes


with


a Li-190SB


(LiCor)


quantum


sensor


(400


nm range)


After


7 d,


the


cultures


with


fungal


or bacterial


contamination


were


discarded.


Contami


nant-free


cultures


were


transferred


to fresh


media


at 30-d


interval


routinely


examined


morphological


develop-


ment.


Unl


ess


otherwi


stated,


experiments


were


per-


formed


a completely


randomized


design.


- a


- J_ ^ -


-- -


ml A


'tt,,,:


,..I


/ II -









production;


effects


of long-term


exposure


to BA


on shoot


production


and


plant


morphology;


and,


genotype


response.


Explant


The


five


explant


types


WEC,


WDC,


SEC,


SDC,


and


EA of cultivar


Florigiant


(FG)


were


cultured


medium


supplemented


with


four


concentrations


of BA,


0.05,


0.5,


and


shoot


induction


and


multiplica-


tion.


This


was


a 5 X4


factorial


experiment


a completely


randomized


design,


and


each


treatment


combination


was


repli-


cated


times.


Cultures


were


scored


at 49 d.


Root


induction


and


growth


vitro-oroduced


plantlets.


Ten


shoots


from


the


various


treatments


were


excised


and


transferred


to medium


plus


NAA


(Atreya


et al., 1984).


The


shoots


were


scored


root


production


at 30 d.


Plantlets


originating


from


the


five


explant


types


with


mm or longer


roots


were


transferred


a soilless


mixture


peat:


perl ite:


1 vermiculite)


that


was


auto-


claved


30 min


at 1.1


cm-2


The


plantlets


were


placed


in a greenhouse


under


shade


and


humidity


8 d and


shade


and


80 to 85%


humidity


for


the


following


42 d.


The


plants


were


grown


to harvest


maturity.


Effect


of BA concentration


on multiple


shoot


production.


Twenty-five


WEC


explants


of FG


were


placed


each


the


media


containing


eight


BA concentration


treat-


ments,


and


After


- a


type.


. J


m


I









Effects


of lonq-term


exposure


to BA.


Whole


embryonated


cotyledon


explants


of FG


were


cultured


160-mL


glass


jars


on both


solid


and


liquid


media


supplemented


with


BA L1I


The


liquid


shake


cultures


contained


isolated


indi


vidual


explants


grown


in 15 mL of medium


with


agitation.


The


210,


explants


and


were


scored


d after


shoot


culture


production


initiation.


at 152,


Thirty


shoots


182,


from


both


liquid


and


solid


media


were


excised


at these


interval


placed


on rooting


medium,


scored


root


production


30 d.


The


plantlets


were


grown


to maturity


the


green-


house.


The


percentage


of pollen


staining


was


determined


as an


indication


of pollen


fertility.


Pollen


stainability


was


determined


two


anthers


three


flowers


from


each


three


plants


grown


from


seed


and


from


three


plants


that


had


originated


from


explants


cultured


on solid


medium


supple-


mented


with


BA L1I


The


two


undehisced


anthers


from


each


flower


were


macerated


a drop


of 1%


ace-


tocarmine


on a slide


viewed


after


30 minm.


The


pollen


each


slide


was


classified


as stained


or unstained


three


random


samples


of 50 grains


each.


Genotype


response.


Whole


embryonated


cotyledon


explants


of 20 genotypes


were


cultured


on medium


with


BA L-1


70 d


, and


then


scored


shoot


regenerability.


a a


rr r


1


A


q


1*









30 d.


The


genotypes


tested


were


from


a seed


collection


maintained


the


University


Florida


(Table


Results


and


Discussion


ExDlant


tvye


Multiple


shoot


regeneration


occurred


from


five


explant


types


on all


four


BA concentration


treatments


(Table


2-2)


The


four


cotyledon


explant


types


expanded


to approximately


twice


their


original


size


and


turned


green


within


to 7 d after


culture


initiation.


The


EA explants


and


the


embryos


of explants


WEC


and


SEC


began


germination


within


8 d.


Multiple


shoots


developed


from


WEC,


and


SEC


explants


within


16 d and


from


WDC


and


SDC


within


35 d.


The


deembryonated


cotyledon


explants,


WDC


and


SDC,


regenerated


shoots


from


and


of the


cultured


explants,


respectively


These


shoots


formed


from


callus


that


arose


the


terminal


portion


of a protuberance.


Thi


protuberance


emerged


from


the


base


of the


cotyledon.


These


shoots


were


initially

structures


deformed

This


et al. (1985)


but


feature


They


also


eventually


was


developed


previously


reported


shoot


into


described


development


normal


Bhatia

from


only


of deembryonated


explants


cultivar


TG-17


tured


on medium


supplemented


with


BA L-1


Illing-


worth,


however,


reported


shoot


regeneration


from


85 and


of whole


and


sectional


deembryonated


cotyledons,


respec-









obtained


study


are


attributed


to BA concentration


and


genotype


effects.


The


percentages


of explants


that


produced


shoots


were


greatest


among


the


WEC,


SEC,


and


EA types


(Table


When


percentages


of responsive


explants


and


numbers


of shoots


produced


per


responsive


explant


were


considered


together,


WEC


was


Among


found


the


four


to be superior

concentrations


to the

of BA,


other

the


explant

number o


types.


f shoots


formed


per


responding


explant


was


maximum


the


level


(Table


2-2) .


Root


induction


growth


of in


vitro-produced


plantlets.


When


the


excised


shoots


were


transferred


to the


auxin-supplemented


plantlets


sistent


medium,


transferred


with


roots


to soil.


report


developed


This


Atreya


and


ability


the


to root


et al. (1984)


esultant

is con-


One


or two


roots


developed


from


the


shoots


that


were


excised


from


the


various


explants


within


14 d of


transfer


to basal


medium


supplemented


with


NAA


Root


growth


occurred


62.5%


of all


the


cultures


within


30 d,


and


approximately


.5 roots


formed


per


responding


shoot.


Of 120


plantlets


transferred


to the


greenhouse,


(92%)


survived


the


tial


50-d


transfer


period.


Thereafter


, 100%


those


plant-


lets


surviving


developed


successfully


, matured,


and


seed,


indicating


that


the


five


explant


types


allow


S a.5 p a 5 aa


q q


I 1


1


* k


1 *








Effect


BA concentration


on multiple


shoot


formation.


Since


the


largest


number


of shoots


arose


from


the


highest


concentration


tested


the


previous


experiment,


WEC


explants


of FG


were


placed


on an expanded


range


of BA


con-


centrations.


After


14 d in


culture,


buds


arising


from


the


plumule


and


cotyledon


tissue


began


to develop


into


multiple


shoots


on the


8 BA concentrations


tested


(Table


2-3) .


Some


the


The


regenerated


remaining


indented


basal


shoots


shoots

region


(23%)


developed


(77%) developed

of the cotyledo


from


directly

n that s


the


from


urrounds


plumule.


the


the


embryo


axis.


Thi


tissue,


which


elongated


to within


and


expanded


to 10


supported


bud


formation


along


adaxial


surface


(Fig.


2-1).


Seventy-two


percent


the


WEC


explants


formed


shoots


on the


from


basal


the


medium,


frequencies


which

found


was


not


among


significantly


the


explants


different


cultured


the


remaining


seven


BA concentrations


(Table


2-3) .


The


incorporation


of BA into


the


medium


therefore


was


not


effec-


tive


promoting


shoot


formation


terms


the


number


explants


responding.


It did,


however,


have


a positive


influence


on the


shoot


organogenesis


of explants


that


did


respond.


The


largest


number


of shoots


produced


per


explant


(9.2


- 9.3)


was


observed


on medium


supplemented


with


BA LI


(Table


2-3) .


Surprisingly,


morphologically


- S a


1


I- 'h *









Plantlets


with


leaves


and


well-developed


root


systems


were


obtained


to 28 d after


placing


the


excised


shoots


from


BA concentration


treatments


on rooting


medium.


Upon


transfer


to a greenhouse,


mature


plants


were


obtained


98 d


(Fig.


-2) .


Effects


of lona-term


exposure


to BA.


The


level


of BA


found


to be optimum


shoot


production


these


experi


ments


were


rather


high


comparison


to the


levels


used


multiple


shoot


induction


other


plant


species


(Flick


al., 1983).


BA L-1


Therefo


concentration


re, the

on shoot


long-term e

production


effects

and m


of the


lorphogenesis


were


tested.


The


use


of liquid


vs.


solid


medium


long-


term


culture


also


was


evaluated.


Thi


level


of BA


allowed


increased


frequency


of shoot


formation


over


time


from


WEC


explants


cultured


both


liquid


and


solid


media


(Table


2-4) .


At 238


d after


culture


initiation,


mean


41.3


and


22.7


shoots


had


regenerated


per


responding


explant


cultured


on solid


(Fig.


and


liquid


(Fig.


media,


respectively.


Solid


production


medium


both


was


superior


terms


to liquid


of numbers


medium


of shoots


shoot


produced


and


because


shoots


that


arose


from


explants


liquid


culture


were


vitreous.


laminae


that


vitro


were


narrow


physiological


anomaly


less chlorophyllous


resulted

, with


a~~~~~~. -naS.


1


* --


1 11









Shoots


were


excised


at 154,


182,


210,


and


d after


culture


initiation,


evaluated,


and


transferred


to rooting


medium.


The


number


of shoots


that


developed


roots


declined


with


increased


culture


age


shoots


originating


on both


liquid


and


obtained


solid


from


media.


liquid


The


was


number


, however,


rooted


much


reduced.


shoots


Approxi


mately


, and


respectively,


of shoots


derived


from


liquid


medium


produced


roots,


as compared


and


of shoots


regenerated


on solid


medium.


Rooted


plantlets


from


culture


ages


transferred


the


greenhouse


developed


and


grew


to maturity


with


no mor-


phological


present


variation.


plants


The


percentages


originating


vitro


of stainable


after


pollen


were


significantly


different


from


those


plants


grown


from


seed.


Means


of 90 and


stainability


were


found


among


the


viewed


pollen


from


vitro-


and


seed-produced


plants,


respec-


tively.


Evaluation


other


aenotvDes


A wide


range


peanut


genotypes


was


evaluated


plant


production


using


thi


regeneration


protocol.


The


range


of shooting


response


among


the


genotypes


was


to 94%


of all


explants


cultured


(Table


-5).


Varying


amounts


of shoots


regenerated


from


the


responding


explants


of each


genotype.


Although


there


was


quantitative


variation


both


shoot


and


root


development


_


I __


_ 1


I









plantlets


from


seed


explants


of peanut.


Optimal 1


quantities


of shoots


are


obtained


with


WEC


explants


cultured


on solidi


field


medium


supplemented


with


BA L-1


Long-term


pro-


lific


plants


cultures


that


can


appear


be produced


to be normal


maintained,


and


resulting


fertile.


Thi


procedure


can


be used


clonal


propagation


and


genetic


transformation


studies


peanut.


In breeding


stud-


les


of seed


nutritional


composition,


these


procedures


would


allow


the


production


multiple


plants


from


one


cotyle-


don,


while


the


other


cotyledon


is utilized


chemical


analyses.














Table


2-1.


Peanut


genotypes


used


seed


explant


cultures.


Genotypet Market type Plant growth habit

Florunner cv. Runner Runner
Sunrunner cv. Runner Runner
Chico cv. Spanish Bunch
NC-7 cv. Virginia Spreading bunch
Florigiant cv. Virginia Runner
392-x ( Virginia Runner
393-7 Virginia Runner
487B Virginia Spreading bunch
435-OL-2 Spanish Bunch
562A Spanish Bunch
803-1 Runner Runner
623B Valencia Bunch
640A Valencia Bunch
558A Spanish Bunch


CV.


= cultivar.


Component


Virginia -
seed kg-1;
seeds (154
with 2-see
with 3- to


lines


of Florigiant.


alternate
Runner a
0 seed kg-
ded pods;
4-seeded


branched form with large seeds (1100
alternate branched form with small
L); Spanish sequential branched form
Valencia sequential branched form
pods.


Runner prostrate growth habit; Bunch
habit occurring in sequential branched
bunch upright growth habit occurring
branched forms.


- upright growth
forms; Spreading
in alternate























>4
R

'0
0)
U

a)
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a)
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Ct
-ri

(0

-' a
4a
o 0>

43
i4-'C




CO
Pr(0
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rd X



oQI a
a



OH
-^ r-i



'-4
rdt


O ^


0 V


I O
S>i


II Q)

M
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Wi

1o 1



'00
Q) d
r-1 <
>4r4
43>
0 -
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u


Sa


- 0


-. &


(L














Tabl


Shoot


production


from


whole


embryonated


cotyledon
varying 1


explants


evels


of Florigiant


peanut


of 6-benzylaminopurine


(BA)


BA No. (%) Mean no. of Difference
concentration responding shoots per from
(mg L-1) explantst responding explant control


(Control)


(72)


(68)


(61)

(71)


(73)


(61)


(61)


Significant
significant


No significant
X2 = 3.09.


the


0.01 probability


differences


among


level


. ns


treatments


= not


at 5%


level















Table


2-4.


Comparison of mean shoot production


from 36


whole embryonated cotyledon explants of
Florigiant peanut on solid and liquid media,


supplemented with 25 mg


(BA)


6-benzylaminopurine


over time.


Days after culture initiation

Media 154 182 210 238


Solid 11.5 16.8 31.5 41.3

Liquid 7.7 10.4 16.8 22.7



Difference 3.8*** 6.3*** 14.7*** 18.5***


Significant at the 0


.001


probability


level.














Table


2-5.


Morphogenic responses of peanut cultivars
culture.


Shoot Productiont


Root Production$


Cultivar


Responding
Explants


NO. per
Responding
Explant


Responding
Shoots


NO. per
Responding
Shoot


Florunner
Sunrunner
Florigiant
Chico


(44)


(70)


NC-7


487-B


(71)


435-OL-2


393-7
392-B
392-C
392-E
392-F
392-G
392-H
392-I
562A
803-1
623B
640A
558A


(69)
(84)
(78)
(65)
(79)


(73)
(94)
(88)
(94)
(50)
(79)


13.1lab
10.1la-d


14.4a


6.6de
7.6cde
6.1de


4.6e


9.3b-e
6. de


7.2cde
5.7de
6.1de
4.6e
5.9de
12.labc
6.1de
8.7b-e
10.3a-d
7.9cde


(85)
(60)
(60)


6.4b-e
7.lbcd
4.5def


1.4f


(65)
(56)
(68)
(42)
(85)
(80)
(75)
(65)
(60)


(65)
(90)
(65)
(85)
(95)
(90)


7.5bcd
4.6def
6.2b-e
3.1ef
11.3a
8.9abc
9.2ab
11.la
7.3bcd
6.5b-e
7.1bcd
4.3def


.Obcd


3.9def


.5bcd


5.3cde


shoots


explants cultured on
weeks.


Production of roots from e:
1-napthaleneacetic acid L-1

Mean separation by Duncan'
level.


from whole embryonated cotyledon
25 mg 6-benzylaminopurine L-1 for


excised shoots placed on 1 mg
for 30 days.


s multiple range test at


Production of








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CHAPTER


ORGANOGENESIS


OF PEANUT


FROM


AND


CULTURED
PERENNIAL


LEAF


TISSUE


PEANUT


Organogenesis


the


process


of regenerating


plants


vitro


from


involves


callus


the


or differentiated


culturing


of explant


tissue.

ssue on


Thi


process


i a defined


medium


containing


an appropriate


hormone


and


nutrient


regime


under


a controlled


of environmental


conditions.


The


cell


grown


culture


divide


and


differentiate


to form


shoots


and/or


roots.


Shoots


which


form


without


roots


generally


are


separated


and


placed


on a medium


favoring


the


development


roots.


In vitro


regeneration


allows


the


potential


produc-


tion

ing


many


traits


plants


desirable


from

for


a single

improved


plant


exhibiting


cultivars.


It al


or carry-


pro-


vides


an alternative


vegetative


propagation


means


crop


plants


that


are


normally


seed


propagated.


One


such


species


perennial


peanut


, Arachis


alabrata


Benth.


which


a tropical


rhizomatous


legume


that


produces


high


quality


forage


that


can


be grazed


, hayed,


made


into


silage


(Prine


et al


, 1981)


is a new


crop


southeastern


U.S.


farmers,


establishment


costs


are


high









A large


number


of plant


species


have


been


regenerated


from


vitro


culture


(Binding


and


Schroeren,


1984),


although


some


of the


more


important


crops,


notably


legumes,


have


lagged


behind


development


of regeneration


tech-


niques.


species


Flick


which


et al.


have


(1983)


been


provides


regenerated


a list


vitro,


of 25 legume


although


most


cases


regeneration


occurred


at low


frequencies.


Little


work


has


been


reported


on peanut,


Arachis


hvDoQaea


but


the


accumulated


literature


from


the


last


seven


years


demon-


states


the


potential


vitro


techniques


improvement.


Mroginski


et al.


(1981)


induced


shoot


regeneration


from


the


callus


immature


leaves


of peanut


CV.


Colorado


Man-


fredi


cultured


on medium


supplemented


with


each


6-benzylaminopurine


(BA)


and


1-napthal eneacetic


acid


(NAA)


and


Pittman


et al.


(1983)


later


extended


thi


study


to 28


genotypes.


All


genotypes


produced


callus,


produced


shoots,


and


produced


roots.


Callus


production


generally


started


on the


basal


end


the


leaflet.


Shoot


meristems


developed


on the


periphery


the


callus


Histological


examination

meristems


the


originate


culture

d from


revealed


adaxial


that


epidermal


embryos


cell


and

near


shoot

the


midrib


the


leaflet.


Pittman


et al.


(1984)


and


Johnson


and


Pittman


(1986)


k8U a a


1


*


r r 1 1


1 II i


I


lil









Cercospora


arachidicola


Hori


and


CercosDoridium


Dersonatum


(Berk.


and


Curt.)


Deighton.


They


found


that


explant


orien-


station


was


important,


with


twice


as many


explants


forming


shoots


when


the


abaxial


epidermis


, rather


than


the


adaxial,


was


contact


with


the


agar


They


also


found


that


the


presence


of midrib


the


explants


reduced


the


number


shoots


formed


per


culture,


and


larger


numbers


of shoots


could


be obtained


increasing


the


light


intensity


to 53


nmol


Bajaj


leaf


et al.


explants


(1981a)


of cultivate


established

d peanut cu


callus


Itured


cultures

on medium


from

sup-


plemented


with


indole-3-acetic


acid


(IAA)


and


kinetin


(KN)


Sukumar


and


Rangasamy


(1984)


studied


the


callus


morphology


and


organogenic


potential


of leaf


explants


from


seven


Arachi


species


and


found


these


explants


to be


variable


response.


The


calli


differed


growth


rate


and


texture,


being


either


smooth


or nodular


and


firm


soft.


the


Three


calli


species

any spe


developed


cies.


roots


and


Narasimhulu


no shoots


and


Reddy


formed


(1983)


reported


sporadic


shoot


development


from


leaf


callus


turned


on medium


supplemented


with


BA LU-


and


NAA


The


tive


above


potential


studi


of leaf


provide

tissue


information

of Arachis s


on the


species


regenera-


cultured


S* *


* I S


*


* a


I A A I


mtl ,


Y~'YL~


a Al 1


rLrl aX 4hlX~i


u~ rLr rL


X









investigates


the


organogenic


capacity


of peanut


leaf


tissue


cultured


on medium


supplemented


with


elevated


concentrations


of cytokinin.


It also


investigates


the


regeneration


response


of 20 peanut


cultivars


and


two


perennial


peanut


cultivars.


Materials


and


Methods


Peanut


ized


CV.


ethyl


Florigiant


alcohol


(FG)


for


seeds


5 min


were


surface


followed


steril-


three


sepa-


rate


immersions


2.6%


sodium


hypochlorite


4 min


each


on a shaker.


Seeds


were


rinsed


three


times


with


sterile


deionized


water.


The


seeds


were


germinated


at 23C


on a


16/8


h light/dark


mm plastic


petri


cycle


on 0.8%


plates.


Difco


After


8 d,


agar


5-mm


water


leaflets


.100

were


excised

medium.


and

The


placed


nutrient


petri

medium


plates


containing


consisted


25 mL of


of Murashige


and


Skoog


major


and


minor


salts


(1962),


sucrose,


mg glycine


nicotinic


acid


, 0.1


mg pyridoxine


hydrochlo-


ride


NAA


, and


0.8%


Difco


agar.


range


of BA


concentrations,


and


, was


tested.


Each


these


four


treatments


was


replicated


times


a com-


pletely


randomized


design.


The


the


media


were


adjusted


to 5.8


with


potassium


hydroxide


or hydrochloric


acid


prior


to adding


the


agar.


The


media


were


autoclaved


20 mmn.


a


, 0.5


1_


1 *J


_









mm from


the


tubes


with


a Li-190SB


(LiCor)


quantum


sensor


(400


nm range).


After


10 d,


the


cultures


with


fungal


or bacterial


contamination


were


discarded.


Contaminant-free


cultures


were


transferred


to fresh


media


at 30-d


interval


and


routinely


examined


morphological


development.


When


buds


arose


they


were


isolated


and


transferred


to 125


x 25


glass


test


tubes,


containing


20 mL of nutrient


medium


sup-


plemented


with


only


BA L-1


to promote


shoot


elongation.


When


shoots


reached


mm they


were


transferred


to the


ini-


tial


culture


medium


containing


NAA


initiate


roots


(Atreya


et al


, 1984).


Plantlets


were


transferred


a 1 peat:


perlite:


vermiculite


greenhouse


potting


medium


that


was


autoclaved


30 min


at 1.1 kg


The


plant-


lets


were


placed


a greenhouse


under


shade


and


high


humidity


40 d and


then


grown


to maturity.


Genotvne


response.


Thirty-five


leaf


explants


from


peanut


genotypes


were


cultured


on nutrient


medium


supple-


mented


with


BA L-1


90 d,


and


then


scored


shoot


and


root


development.


The


genotypes


tested


were


from


a seed


collection


maintained


the


University


of Florida


(Table


Perennial


peanut.


Leaf


explants


from


two


perennial


peanut

on medi


cultivars, 'Fl

a supplemented


origraze


with


' and


NAA


'Arbrook


plus


were


the


cultured

concen-


2' -.


-- -i


-- r. .t~


_-___ A


S-


L-~--LI. -~-LI


CI ----


I








sterilized


hypochlorite


ethyl


10 min.


alcohol


Explants


5 min


were


and


rinsed


2.6%


three


sodium


times


with


sterile


deionized


water


Sixty


explants


from


each


tivar


were


cultured


on each


BA concentration


treatment,


and


evaluated


90 d after


culture


initiation.


Results


and


Discuss


Within


six


days


of culture


initiation,


FG leaflets


began


to expand


and


thicken


with


media


treatments.


production


and


organogenesis


occurred


on all


four


concentration


treatments


(Table


3-2) .


Callus


developed


from


the


adaxial


surface


within


21 d,


while


bud


issue


developed


from


the


adaxial


surface


36 d after


culture


initiation


(Fig


-1).


Roots


developed


from


the


callus


within


45 d.


Ninety-six


percent


the


explants


developed


callus


the


BA 1


treatment,


which


was


not


significantly


dif-


ferent


from


the


frequencies


found


among


the


leaflets


tured


on the


remaining


three


treatment


level


The


concen-


tration


of BA did,


however,


have


a significant


influence


the


capacity


explants


to produce


organs.


The


largest


number


of explants


that


developed


bud


tissue


(38%)


was


observed


on medium


supplemented


with


BA L~-


. This


num-


ber


much


lower


than


that


obtained


Mroginski


et al.


(1981) ,


tured


who


leaf


reported


explants


bud


of peanut


tissue


CV.


development


Colorado


from


Manfredi.


a-i


I r -


_









declined,


with


no roots


formed


at a concentration


of 10


These


results


are


comparable


to the


rooting


results


reported


Mroginski


et al.


(1981).


The


formation


both


bud


tissue


and


roots


from


a single


explant


was


not


observed.


Isolated


bud


tissue


was


then


tran


sferred


to basal


medium


supplemented


with


BA L-I


to promote


shoot


dif-


ferentiation


and


elongation,


and


percent


these


tures


developed


one


to three


shoots


(Fig.


within


transfer


When


these


shoots


were


transferred


to the


auxin-supplemented


medium,


100%


formed


plantlets


with


one


two


roots


within


35 d,


were


transferred


to soil.


The


capacity


of shoots


regenerated


from


peanut


leaf


tissue


root


upon


transfer


to medium


supplemented


with


NAA


was


previously


described


Narasimhulu


and


Reddy


(1983)


Of 27 plantlets


tran


sferred


to the


greenhouse,


(85%)


survived


the


initial


40-d


transfer


period


and


developed


successfully


to maturity


No phenotypic


variants


were


observed


the


regenerants.


Evaluation


other


cenotvpes.


A wide


range


of peanut


genotypes


was


evaluated


organogenic


responsiveness.


Although


genotypes


produced


callus


similar


frequen-


cies,


significant


differences


bud


and


root


formation


occurred


among


those


tested


(Table


The


majority


cultivars


developed


bud


tissue


during


the


90-day


culture


_a_ __ -


*.


-I


.. 1*








genotypes.


Much


variation


callus


production


and


organogenesis


was


noted.


Perennial


peanut.


Within


30 d of culture


initiation,


of the


leaf


explants


formed


callus.


The


number


explants


of Arbrook


and


Florigraze


which


formed


callus


was


not


significantly


different


among


the


four


treatments.


50 d,


dense


friable


callus


containing


many


shoot


meristems


developed


from


the


explants


(Fig.


3-3) .


The


concentration


of BA had


a positive


influence


on the


number


of calli


yield-


meristems


(Table


-4) .


The


large


st number


of cultures


forming


and


continued


eristems

BA L-1.

growth


was


observed


A small


and


on media


quantity


differentiation


supplemented


these m

towards


leristen

shoot


with


(10%)


develop-


ment


(Fig.


3-4)


upon


transfer


to basal


medium


supplemented


with


BA L-1


, although


additional


studies


are


required


to continue


this


protocol


complete


plant


regeneration.















Table


3-1.


Peanut


genotypes


used


leaflet


cultures.


Genotypet Market type Plant growth habit

Florunner cv. Runner Runner
Sunrunner cv. Runner Runner
Chico cv. Spanish Bunch
NC-7 cv. Virginia Spreading bunch
Florigiant cv. Virginia Runner
392-x Virginia Runner
393-7 Virginia Runner
487B Virginia Spreading bunch
435-OL-2 Spanish Bunch
562A Spanish Bunch
803-1 Runner Runner
623B Valencia Bunch
640A Valencia Bunch
558A Spanish Bunch


CV.


= cultivar.


Component


Virginia
seed kg-1
seeds (1!
with 2-se
with 3-


lines


of Florigiant.


- alternate
SRunner a
40 seed kg-
reded pods;
.o 4-seeded


branched form with large seeds (1100
alternate branched form with small
L); Spanish sequential branched form
Valencia sequential branched form
pods.


Runner prostrate growth habit; Bunch
habit occurring in sequential branched
bunch upright growth habit occurring
branched forms.


- upright growth
forms; Spreading
in alternate















Table


Morphogenic
explants to
aminopurine


response
varying
(BA) .


of Florigiant
concentrations


peanut leaf
of 6-benzyl


responding


explants


Concentration


Callus


Bud


Root


Development


43(96)


Formations


2(4)


Formation


16(35


44(98)


4(9)


43(96)

42(94)


17(38)


3(7)


9(20)


0o(o)


NS = No significant
level.


differences


the


0.05 probability


The


least


significant


difference


(LSD)


test


values


comparing


two


BA concentrations


are


21 at


the


level


of significance


and


the


level


The
are


LSD test
20 at the


values


comparing


level


two


significance


BA concentrations


and


26 at


the


level















Table 3-3.


Morphogenic response of leaf


explants of peanut


cultivars


in culture.


responding explantst


Callus


Bud


Root


Cultivar


Development$


Chico


Florigiant
Florunner
Sunrunner


562A
558A
623B
640A


487B
393-7


435-OL-2


NC-7


392-B
392-C
392-E
392-F
392-G
392-H
392-I


30(93)
33(100)
33(98)
30(100)
35(100)
32(96)
31(97)
34(96)
30(94)
33(93)
34(96)
35(100)
34(100)
31(92)
33(93)
31(92)
30(94)
33(93)
35(100)
35(100)


Formation


5(15)
7(20)
6(20)


4(14)
2(5)
6(19)


4(12)
2(5)
2(6)
9(26)


4(10)
3(9)
5(15)


Formation


2(7)
3(10)
4(13)


1(4)


4(12)
2(6)


4(11)


Leaf


explants were cultured on medium supplemented with


6-benzylaminopurine L'-


for 90 days.


NS = No significant differences at
level.


Significant at the


Significant at


the


0.005 probability


0.005 probability


0.05


level,


level,


probability


= 44.89.


= 42.87.














Table


3-4.


Morphogenic response of leaf explants from
perennial peanut cultivars Arbrook and Flori-
graze to varying concentrations of 6-benzyl-
aminopurine (BA).


BA No. (%) calli with shoot meristemst
Concentration
(mg Ll ) Arbrook$ Florigraze$


1 15(27) 10(17)

3 32(59) 26(47)

5 30(57) 24(41)

10 19(36) 17(32)


Percent response
initiation.


was


determined


90 days


after


culture


The least significant
two BA concentrations
level of significance


difference test values
for each cultivar are
and 30 at the 1%.


for comparing
23 at the 5%







37





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0 -















CHAPTER


IN VITRO


CULTURE


OF PEANUT


ANTHERS


The


tissue


culture


of anthers


a means


of obtaining


haploid


plants,


which


are


plants


possessing


the


gametophytic


number


of chromosomes


in their


sporophytes,


from


micro-


spores.


The


technique


involves


plating


anthers


at the


pro-


per


developmental


stage


on a defined


medium.


Uninucleate


microspores


before


or during


mitos


are


most


inductive


towards embryo

dissected out

directly onto

procedures are


development (

of unopened, s

an appropriate


adjusted


Bajaj


, 1983)


terilized

agar med


so that


the


The


flower


ium.


anthers


buds


The


microspores


and


medium

are i


are

plated

and


nduced


to divide.


Their


division


results


either


direct


develop-


ment


into


embryos


through


stages


analogous


those


nor-


mal


diploid


seed


embryos


or the


production


an undifferen-


tiated


callus,


which


when


transferred


to a regeneration


medium


will


differentiate


shoots


roots


(Reinert


and


Bajaj,


1977).


Plants


regenerated


from


anther


culture


will


often


have


variation


ploidy


level


Nonhaploid


plants


can


conceiv-


ably


from


microspores,


polyploidization


of pollen


callus


cells,


or from


callus


derived


from


somatic


tissue









Guha


and


Maheshwari


(1964)


first


reported


haploid


plant


production


from


the


pollen


of Datura


inoxia


Mill.,


and


since


that


time


anther


culture


has


been


tried


with


a number


economic


species


(Bajaj ,


1983)


technique


valuable


because


the


haploid


production


plant


development


of homozygous


offers


plants.


The


a rapid

y also


means


can


facili


tate


the


detection


of useful


traits,


unique


recombinants,


and


mutations


because


they


possess


only


one


allele


at each


locus


They


also


may


allow


wide


hybridizations


with


species


having


similar


chromosome


numbers


(Bajaj ,


1984).


With


Arachi


anther


culture


progress


has


been


limited


the


production


of nonhaploid


plants


(Bajaj


et al.,


1981b,


Mroginski


and


Fernandez,


1980,


and


Seitz


et al.,


1985)


and


the


production


of multicellular


microspores,


an early


stage


of embryogenesis


(Bajaj


et al.,


1980


1981b


and


Mroginski


and


Fernandez,


1979).


Bajaj


pollen


et al.


embryogene


(1980,

sis in


1981b)

anther


reported

cultures


the


induction


of Arachis


villosa


Benth.


and


Arachi


alabrata


Benth.


the


first


study


they


cultured


anthers


of A.


alabrata


and


peanut,


Arachis


hvpoqaea


the


uninucleate


and


binucleate


microspore


stages


Murashige


and


Skoog


medium


supplemented


with


indole-3-


acetic

spores


acid

of A.


(IAA)


qlabrata


and


divided


kinetin

to form


(KN)


embryos


The


and,


micro-


some


-I S a --


-r


II 1~


1


I









same


medium


when


microspores


were


undergoing


mitosi


Callus


developed


within


21 d from


and


the


anthers,


respectively


and


only


microspores


of A.


villosa


were


reported


to have


undergone


repeated


nuclear


and


cell


divisions


to form


embryos.


These


microspores


prolifer-


ated


to form


callus


which


was


more


compact


than


callus


obtained


from


anther


tissue.


Within


the


two


callus


types


could


not


be distinguished


from


each


other.


The


calli


were


transferred


leneacetic


acid


to medium


(NAA)


supplemented


and


with


mg 1-naptha-


mg 6-benzylaminopurine


(BA)


and


began


to form


organs


within


Eighteen


percent


the


calli


from


M 13 produced


roots


with


occasional


shoot


formation,


and


the


calli


from


villosa


different


ated


shoots


which


were


transferred


to medium


supplemented


with


NAA


rooting


was


reported


whether


these


organs


differentiated


from


microspore-derived


callus


or from


anther


tissue-derived


callus.


The


chromosome


numbers


of M 13 callus


cell


showed


a range


of variation


from


20 to 80,


while


the


chromosome


numbers


of A.


villosa


callus


cell


were


reported.


Mroginski


peanut


and


. Colorado


Fernandez

Manfredi,


(1979) c

Arachis


ultured


anthers


correntina


(Burk)


Krap.


et Greg.,


villosa


on medium


supplemented


with


NAA


and


BA 1-1


Callus


was


produced


from


60%,


- a


C-,- --N '- -A-I -- -- ....t4 -- -- -----------------------------------


- .C A.. 1~ -


mt.-


*!,,,,


q


,I A -


u-


Cn_ n r- J J n









other


hand,


anthers


of A.


correntina


and


villosa,


both


2n=20,


yielded


callus


cell


with


10 chromosomes.


Multicel


lular


microspores


were


found.


These


workers


(1980)


cultured


anthers


of Arachi


lianosa


(Chod


. et


Hassl.)


Krap.


et Greg

medium,


. at

and


the


uninucleate


approximately


microspore


the


stage


anthers


on the same

developed c


lus from


somatic


tissue


within


30 d.


Plantlets


developed


these


calli


when


transferred


to medium


with


0.01


BA L


Martin


and


Rabechault


(1976)


cultured


stamens


of peanut


on medium


supplemented


with


0.46


mg 2,4-dichlorophenoxy-


acetic


acid


,4-D)


, 0.4


NAA


, and


0.45


KN L-1


Microspore


divi


sion


was


arrested


as soon


as the


stamens


were


cultured.

filament


These

within


anther

15 d.


produced


Pittman


callus


(1981)


the


cultured


base


anthers


the

con-


training


microspores


the


late


tetrad


stage


of development


from


10 genotypes


representing


seven


Arachis


species


They


were


cultured


on solid


medium


supplemented


with


1.86


NAA


and


0.02


KN L~1


and


liquid


medium


with


NAA


and


BA L-1


float


cultures.


The


anthers


produced


callus


both


cultures


the


site


where


the


filaments


were


removed.


Seitz


et al.


(1985)


isolated


a highly


morphogenic


line


from


the


anthers


of Arachi


paracuariensis


Benth.


- a -- 1- --


L


_I _


I r-


I _


I









remnants


both


systems


and


supported


shoot


development


within


to 10 wk of culture


initiation.


These


shoots


regenerated


roots


upon


transfer


to medium


supplemented


with


NAA


In conclusion,


success


with


anther


culture


peanut


has


been


limited.


Thi


limited


success


may


be due


geno-


type


and/or


media


constraints.


Haploid


callus


cell


of M 13


peanut


were


found


the


anther


cultures


one


study


(Bajaj


et al.,


1981b) ,


however,


another


study


the


microspores


Colorado


Manfredi


were


found


to degenerate


(Mroginski


and


Fernandez,


1979).


Both


studies


used


media


which


suc-


cess


fully


induced


microspore


divi


sion


in the


species


villosa.


Therefore,


genotype


may


play


a greater


role


microspore


divi


sion


induction


than


medium


components.


Embryogenesis


was


induced


cultures


of two


diploids,


villosa


and


correntina,


(Bajaj


et al.,


1981b,


Mroginski


and


Fernandez,


1979)


and


one


tetraploid,


alabrata


(Bajaj


et al., 1980)


In each


case


microspores


under-


went


repeated


divi


sons


resulting


formation


of both


multicellular


microspores


and


callus.


This


chapter


investigated


the


culture


of anthers


from


three


peanut


cultivars.


The


cultures


were


evaluated


the


formation


of embryos


and/or


callus


from


microspores


, and


subsequent


organ


differentiation.


The


media


used


A.,,a "


a U... Wa-, ~ nftL.1- I -2 a 1' -


flu.--


.a


r. i


/ -i f\ il-\


r


;I


L









microspore


stage


to determine


the


proper


flower


bud


sizes


for


culture,


follow


the


survival


and


developmental


course


of microspores


over


time,


and


assess


the


mor-


phogenic


response


the


three


cultivars.


Material


and


Methods


Flower


buds


of peanut


cv.


Florigiant


(FG),


Florunner


(FR) ,


and


Chico


(CH)


were


staged


to establish


a relationship


between


microsporogenesis


and


bud


length.


Flower


buds


various


zes


from


four


greenhou


se-grown


plants


of each


tivar


were


collected


before


0800


Bud


length


was


deter-


mined,


using


a di


sse


acting


microscope,


from


the


base


the


tip


the


sepals.


Four


oblong


anthers


from


each


three


buds


the


same


size


were


evaluated.


A total


of 96,


and


anthers


from


and


respectively,


were


stud-


ied.


The


four


oblong


anthers


from


each


bud


were


macerated


individually


a drop


of 1% acetocarmine


on a slide.


Sixty


microspores


from


each


anther


were


viewed


under


200X


magnifi


cation


after


30 min


and


classified


according


microspore


development


stage.


Microspore


development.


The


survival


and


development


of microspores


over


time


was


followed


FG by


viewing


the


microspores


embryo

cleate


interval


induction medium.

microspores were


from


Oblong

surface


anthers


anthers c

sterilized


cultured

ontainin


on an

g uninu-

ethyl


C~~~ S


,r ii


1


_


A


1


A


1 -












of medium.


The


nutrient


medium


consisted


of Murashige


and


Skoog


s major


and


minor


salts


(1962),


sucrose,


mg gly-


cine


nicotinic


acid


mg pyridoxine


, and


and


Difco


KN L-1


agar.


(Bajaj


was


et al.,


supplemented


1981b).


The


with


IAA


the


medium


was


adjusted


to 5.8


with


potassium


hydroxide


prior


adding


the


agar.


The


medium


was


autoclaved


at 1


.1 kg


cm-2


20 min.


light/dark


Cultures


cycle.


were


General


maintained


Electric


at 23C


F40CWRS.WM


on a 16/8


fluorescent


tubes


were


used


which


produced


approximately


90 pmol


when


measured


mm from


the


tubes


with


a Li-190SB


(LiCor)


quantum


sensor


(400


nm range)


Microspore


observa-


tions


were


made


d after


culture


initiation


and


thereafter


7-d


interval


70 d


Three


anthers


were


removed


from


culture,


macerated


acetocarmine,


and


viewed


individu-


ally


microspore


development.


Morphoqenic


response.


Oblong


anthers


containing


uninu-


create


microspores


of FG,


and


were


surface


sterilized


and


cultured


on the


agar


solidified


nutrient


medium


supple-


mented


with


either


NAA


and


BA LI1


(Medium


as used


Mroginski


and


Fernandez


(1979),


or 4


and


KN L~-


(Medium


as used


Bajaj


et al.


(1981b)


After


tion


the


were


cultures


discarded.


with


Cultures


fungal


were


or bacterial


transferred


contamina-


to fresh


-L- -- -, A -2


0.8%


J


1


7


- ..r ^ _


--


F









with


either


0.01


BA L1I


(Mroginski


and


Fernandez,


1979),


NAA


and


BA L'-


(Bajaj


et al.,


1981b),


respectively,


to initiate


organogenesis.


Ninety


anthers


each


cultivar


were


cultured


a completely


randomized


design.


Results


and


Discussion


Four


oblong


anthers


from


each


of three


flower


buds


the


same


size


were


stained


acetocarmine


assess


the


stage


of development


the


microspores.


Microspore


devel


opment


was


synchronous;


the


anther


samples


of each


bud


size


of FG,


and


CH consistently


contained


97 to 100%


the


total


number


of microspores


the


same


stage


of devel


opment.


Table


presents


relationship


between


the


flower


bud


lengths


and


the


stage


of development


the


microspores


the


three


cultivars.


Buds,


to 2


to 3.5


tively,


and


contained


to 1.5


mm long,


microspores


of FG,


which


were


and


respec-


uninucleate.


Cur-


rent


knowledge


indicates


that


microspores


the


uninucleate


stage


are


most


inductive


towards


embryogenesis


(Bajaj,


1983) .


Anther


cultured


the


following


experiments


were


obtained


from


flower


buds


of these


sizes.


Microsnore


development.


Acetocarmine


preparations


FG anthers


showed


that


4 d after


culture


initiation


the


maj ority


of microspores


remained


unchanged.


Comparatively,


SS --


or 1


-- 1


R *


- 1


.









microspores


were


shriveled,


and


the


remaining


were


lightly


stained


ated,


uninucleate


were


(Fig.


uninucleate,


-1) .


At 28 d 50%


and


were


had


binucleate


degener-


(Table


-2) .


An interesting


aspect


of the


binucleate


microspores


was


a differential


response


to staining


acetocarmine.


one


group


the


cytoplasm


stained


darkly,


partially


obscuring


the


two


nuclei


(Fig.


-2).


In the


other


group


the


cytoplasm


stained

allowing


lightly,


the


indicating


microspore


a lower


segmentation


star

and


content


nuclei


and


to be easily


viewed


(Fig


Thi


double


response


phenomenon


has


been


reported


tobacco


Sunderland


Wicks


(1969)


They


found


that


densely


staining


type


developed


subsequently


like


normal


pollen


and


that


the


lightly


staining


type


proved


to be embryoidal


They


observed


both


binucleate


microspore


types


2 d after


culture


initiation.


Mitosis


occurred


the


embryoidal


type


on the


d of culture,


and


multicellular


embryos


were


observed


on the


10th


Further


staining


development


binucleate


both


microspores


the


lightly


of FG did


not


and


densely


occur


during


70-d


observation


period.


Similar


quantities


of degener-


ated, uninucleate,

staining binucleate


densely


staining


microspores


were


binucleate,

observed a


and


lightly


it each


the


remaining


observations


(Table


4-2).


Morphoqenic


response.


The


exci


anthers


of FG,


-- .


__ I


1


q


I


dK


I









cases,


callus


first


appeared


at either


the


cut


end


the


stamen


filament


or on the


anther


wall.


A higher


percentage


of responding


anthers


cultured


on Medium


M than


on B for


each


cultivar


was


noted


(Table


-3).


When


callus


was


trans-


ferred


the


organ


differentiation


media,


roots


began


differentiate


FG cultures


within


11 d.


The


formation


roots


provides


evidence


for


morphogenesis


and


possible


plantlet


regeneration


Organogenesis


reported


from


Bajaj


from


the c

et al.


somatic


:allus


anther


of peanut


(1981b),


who


tissue


anthers


observed


of FG.


was

root


first

develop-


ment


. M 13


cultures.


In a few


instances,


callus


formation


was


initiated


within


the


anthers


and


burst


through


the


wall


as a mass


approximately


mm in


diameter


(Fig.


This


formation


occurred


and


0 of


the


CH, FG,


and


FR anthers,


respectively,


and


was


observed


only


on Medium


14 and


20 d


after


culture


initiation


FG and


respectively.


vegetative


structure


resembling


a shoot


formed


from


this


callus


a culture


of FG


(Fig.


and


(Fig.


within


34 d of culture


initiation


on Medium


(Table


-3).


Subsequently,


the


callus


and


structures


ceased


development,


turned


brown,


and


died


within


6 d of


transfer


to medium


supplemented


with


0.01


BA L-1


In conclusion,


varying


flower


bud


sizes


the


three


-l a


r









Many


of the


microspores


degenerated


within


28 d of culture,


while


others


remained


uninucleate.


Mito


commenced


the


14th


d of culture,


with


approximately


the


uninucleate


microspores


completing


their


first


divi


sion


28 d.


evidence


of further


embryo


or pollen


development


was


noted


Although


plantlet


development


from


anthers


was


not


observed,


root


organogenesis


occurred


from


the


callus


of somatic


anther

that o


tissue


originate


of FG.

d within


Shoot


structures


an anther


of both


merged

FG and


from

CH,


callus

however,


no further


development


occurred.





















Table


4-1.


Relationship


between


microsporogenesis


flower
three


bud
peanu


length


and


cult ivars.


Microspore
development
stage


Flower


Florigiant


bud


length,


Florunner


Chico


Tetrad


<1.0


Uninucleate


1.5-2


2.0-3


1.0-1


Binucleate


.5-3.5


.0-4.5


2.0-3.0


Mature


pollen


-4.0


Bud
the


length
tip of


was
the


determined


measuring


from


the


base


sepals.















Tabl


Microspore


survival


and


development


of Flori


giant


peanut


over


time.


microspore


stage


Days


Densely
Stained


Lightly
Stained


Culture$


Degenerated


Uninucleate


Binucleate


Binucleate


5(8)


55(9


45(75


21(35)


27(45


10(17)


17(28)


29(48)


10(17)


4(7)


30(50)


12(20)


6(10)


11(18)


4(7)


30(50)


5(8)


28(47)


6(10)


29(48)


6(10)


28(47)


29(48)


12(20)


6(10)


Each
from


value


each


represents


three


the


anthers


mean


response


macerated


of 60 microspores


individually


acetocarmine.


Anthers
mented
kinetin


were


with


cultured


on agar


indole-3-acetic


solidified


acid


medium


supple-


and















Table


In vitro
anthers


morphological


of three


peanut


responses


of excised


cultivars.


No.(%) of No.(%) No. forming
Cultivart callusing anthers forming roots* shoots


Chico (B) 10(23)* 0 0

Chico (M) 19(44) 0 1

Florunner (B) 8(19) 0 0

Florunner (M) 30(68) 0 0

Florigiant (B) 16(38) 5(15) 0

Florigiant (M) 41(93) 19(23) 1


* Mean
the


no.
M and


of responding


B media


anthers


the


of each


cu


0.05 probability


Itivar
level


differ
based


for
on


t-tests.


A total
70 days
acetic


of 90 anthers


on media


acid


leneacetic


'-1
acid


of each


supplemented


and


cultivar


with


were


either


kinetin


(NAA)


and


or 2


L-1
mg


cultured


indole-3-


mg 1-naptha-


6-benzylaminopurine


(BA)


(M) .


Cultures


that


initiation


either
(M), r


tions


were
NAA


respectively,


were


taken


developed


callus


transferred


and


to initiate


30 days


after


within


to media
BA L-1 (I


35 days of
supplemented
or 0.01 ma


organogenesis.
transfer.


culture
with
BA -17


Observa-


Shoot
medium


structures
i 34 days a


developed


afterr


culture


on the


M callus


induction


initiation.








55




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CHAPTER


SOMATIC


EMBRYOGENESIS


FROM


IMMATURE


COTYLEDON


TISSUE


OF PEANUT


Somatic


embryogenesis


the


initiation


and


development


of embryos


from


somatic


tissue


(Ammirato,


1983)


Haberlandt


(1902)


reported


that


somatic


cell


are


totipotent


and


possess


ent


the


(Tisserat


ability t

et al.,


:o produce

1979).


plants

He also


identical

predicted


the


that


par-


thi


phenomenon


would


become


apparent


tissue


cultures


of vari


ous


plant


species.


A somatic


embryo


the


true


manifesta-


tion


of cell


totipotency


It displays


bipolar


development


with


shoot


and


root


produced


as a structure


which


follows


the


developmental


pathway


the


zygotic


embryo.


Somatic


embryos


develop


two


ways


(Sharp


et al.,


1980) .

arise


The


first


directly


way


from


is direct


plant


tissue.


embryogenesis


These


where


embryos


embryos


initiate


from


cell


of explant


material


placed


culture


conditions


that


induce


such


development


(Tisserat


et al.,


1979).


The


sec


method


indirect


embryogenesis


where


cell prolifer-


ation


a prerequis


embryo


formation


(Sharp


et al


1980).


The


cell


the


tissue


differentiate


to form


callus,


and


are


then


induced


toward


embryo


development


A A









Somatic


embryogenesis


was


first


recognized


Steward


et al.


rot.


(1958)


Since


and


then,


Reinert


(1958)


investigations


tissue


have


been


cultures


widespread


car-


and


number


of plant


species


have


produced


embryos


culture


(Tisserat


et al


, 1979)


Although


a variety


of monocots


and


dicots


have


produced


somatic


embryos


culture,


attempts


with


large


-seeded


legumes


have


been


unsuccessful


until


recently


Somati


embryo


induction


in soybean,


Glycine


max


Merrill,


(Barwale


et al


, 1986,


Christianson


et al


1983,


Lippman,


Gamborg


1984,


et al., 1983,


Phillips


Kerns


et al


Collins,


1985,


1981) ,


pea,


Lippman


Pisum


sativum


, (Jacobsen


Kysely,


1984),


and


bean,


Phaseolus


vulaaris


, (Martins


Sondahl,


1984)


were


first


reported


the


1980s


Further


studies


have


revealed


the


direct


and


indirect


production


of somati


embryos


from


zygotic


embryos


of soybean


, 1985)


(Laz


and


zeri


et al


of broad


, 1985,


bean,


cia


et al.,


faba


1985,


(Griga


Ranch


et al


1987)


embryos


The


production


of peanut,


of somati


Arachis


embryos


hvpooaea


has


from


not


the


yet


zygotic


been


accomplished.


Griga


et al. (1987)


produced


somati


embryos


callus


and suspension

from immature


cultures


seed


of broad


cotyledon


bean.


tissue


Callus


cultured


was


induced


on medium


con-


training


,4-dichlorophenoxyacetic


acid


,4-D)


i









Lazzeri


(1985)


regenerated


whole


plants


from


somatic


embryos


derived


from


immature


zygotic


embryos


soybean.


Immature


embryos


were


isolated


from


seed


mm long


and


cultured


on media


supplemented


with


either


mg 2,4


or 10


l-napthaleneacetic


acid


(NAA)


Somatic


embryos


developed


on the


cotyledon


surfaces


within


30 d,


with


higher


frequencies


of embryogenesis


induced


with


than


with


NAA.


However,


embryos


induced


2,4-D


had


abnor-


mal


morphology,


embryos


were


whereas


removed


those


from


induced


the


NAA


cotyledon


were


normal.


surfaces


and


transferred


to medium


containing


0.15


NAA


and


0.03


each


of 6-benzylaminopurine


(BA),


kinetin


(KN),


and


zeatin


to stimulate


shoot


development.


The


plantlets


were


then


transferred


to medium


with


0.005


indole-3-


butyric


acid


(IBA)


to stimulate


root


development.


the


plants


developed


root


systems,


they


were


transferred


pots


containing


autoclaved


soil


and


grown


to mature


plants


bearing


seed.


Ranch


et al. (1985)


regenerated


plants


from


the


imma-


ture


embryo-derived


callus


of 14 soybean


genotypes.


Whole


embryos,


embryo


axes,


and


cotyledons


were


cultured


on medium


supplemented


with


2,4-D


. In 3


wk somatic


embryos


developed


with


varying


frequencies


on both


isolated


cotyle-


dons


and


cotyledons


of whole


embryos


from


14 genotypes.


_









stimulate


apical


development.


Within


30 d the


mature


embryos


had


developed


into


plants


with


three


nodes.


et al.


(1985)


regenerated


somatic


embryos


and


plant-


lets


from


cold-treated


immature


soybean


embryo


fragments.


The


embryos


were


treated,


cut


into


2-mm


segments,


and


then


cultured


on medium


supplemented


with


mg 2,4-D


40 d.


Callus


formed


from


of the


fragments


and


was


transferred


to liquid


medium


containing


mg 2,4


-D L-1


and


coconut


milk,


and


was


incubated


on a rotary


shaker


at 100


rpm


for


14 d.


The


cell


were


then


filtered


and


transferred


at 20 ML


aliquots


to petri


plate


covers


to form


hanging


drop


cultures


were


development


transferred


and


into


maintained


roembryos.

on solid


These

medium


proembryos

containing


BA L-1


and


0.01


indole-3-acetic


acid


(IAA)


for


40 d


to induce


development


into


globular


and


heart-shaped


embryos.


These


were


then


returned


to liquid


medium


supple-


mented


with


BA L~1


and


0.01


IAA


for


different


ation


into


plantlets.


summary,


somatic


embryos


were


derived


from


the


iso-


lated


cotyledons,


immature


soybea


whole e

n seed.


embryos,

The e


and


-mbryo


fragmented

s developed


embryo

from


parts

coty-


ledon


issue


either


directly


or indirectly


from


callus.


Auxins


2,4-D


and


NAA


both


were


effective


embryo


initia-


tion.


Although


embryogenic


cultures


were


derived


from









Thi


chapter


investigated


the


somatic


embryogenic


response


from


the


zygotic


embryo


cotyledons


of three


peanut


genotypes


cultured


on media


supplemented


with


a variety


growth


regulator


treatments.


Materials


and


Methods


Immature


pods


three


peanut


cultivars,


Florigiant


(FG) ,


Florunner


(FR) ,


and


Chico


(CH) ,


were


soaked


water


containing


0.5%


antimicrobial


P-chloro-M-Xylenol


2 h.


Seeds


were


excised


from


the


pods,


and


embryos


ranging


size


from


to 9


mm were


isolated


from


the


seed


coats


The


two


cotyledons


of each


embryo


were


separated


and


the


embryo


axes


removed.


Cotyledons


were


surface


sterilized


ethyl


alcohol


5 min


followed


three


separate


immer-


sions


2.6%


sodium


hypochlorite


4 min


each


on a


shaker.

deionized


Cotyledon

water.


were


rinsed


Cotyledons


were


three


times


placed


with


into


sterile


X 15


plastic


petri


plates


containing


25 mL of medium.


The


nutri-


ent


medium


consisted


of Murashige


and


Skoog


salts


(1962),


sucrose,


glycine


, 0.5


nicotini


acid


, 0.1


pyridoxine


hydrochloride


, and


0.8%


Difco


agar.


Basal


medium


and


a range


of growth


regulator


treatments,


0.01,


0.1,


and


, 5,


of 2,4-D


and


NAA,


and


BA L-1


were


prepared


as treatments.


The


12 media


were


adjusted


5.8 with


potassium


hydroxide


or hydrochloric


acid


and


_ r


>


I









Cultures


were


maintained


at 23C


with


a 16 h daylength.


General


Electric


F40CW-RSeWM


fluorescent


tubes


were


used


which


produced


approximately


85 jmol


when


measured


mm from


the


tubes


with


a Li


-190SB


(LiCor)


quantum


sensor


(400-700


nm range).


After


10 d,


the


cultures


with


fungal


bacterial


contamination


were


discarded.


Cultures


were


transferred


to fresh


media


the


same


composition


at 30-d


intervals


Results


and


Discussion


Callus


formation


occurred


from


only


one


the


1,728


cotyledons


cultured.


A cotyledon


of Chico


cultured


medium


supplemented


d after


culture


with


2,4-D


initiation.


The


developed


callus


callus


originated


within


the


explant


and


emerged


through


the


abaxial


surface.


It continued


grow


on subcultures


to medium


the


same


composition


and


to medium


supplemented


with


BA L-


and


no auxin,


but


did


not


regenerate


during


the


subsequent


period.


Comparatively,


studies


with


soybean


report


direct


development


somatic


embryos,


without


callus


formation,


from


zygotic


embryo


cotyledons


within


only


to 6 wk of cul-


ture


initiation


(Lazzeri


et al.,


1985,


Ranch


et al.,


1985).


There


was


no embryogenic


response


among


the


and


remaining


CH cotyledons


cultured


on the


12 media


treatments


for


The


majority


of peanut


cotyledons


remained


white









few


days


of culture


initiation,


and


unresponsive


cotyledons


remain


white


or turn


brown


culture


(Lazzeri


et al.,


1985,


Ranch


et al


, 1985).


The


regenerat


require


possibility

ion from im


further


of using


mature


research


cotyledon


to determine


procedure

tissue

e the in


plant


of peanut


fluence


will

geno-


type.


Ranch


et al.


(1985)


evaluated


soybean


genotypes


found


that


although


there


was


variation


the


frequency


of somatic


embryogenesis


, all


did


produce


embryos.


The


role


of other


factors


such


as growth


regulator


regime,


tissue


age,


environmental


conditions


also


may


require


further


evaluation.















CHAPTER


SUMMARY


AND


CONCLUSIONS


Success


in peanut,


Arachis


hvpoaaea


improvement


programs


through


the


use


of in


vitro


techniques


will


require


a knowledge


standing


about


for


capacity


requirements


to regenerate


culture.


and


In vitro


an under-


morpho-


genesis


occurred


in a variety


tissue


cultures


from


a num-


ber


of peanut


genotypes


investigated


thi


study.


The


regeneration


response


of explants


taken


from


the


seed of peanut


provides


an efficient


system


for


successful


high


frequency


in vitro


regeneration


of whole


plants.


Plant


regeneration


from


cotyledon


explants


has


been


reported


peanut


(Illingworth,


1968,


1974,


Atreya


et al.,


1984,


Bhatia


et al., 1985)


and


also


other


legumes


(Oswald


et al.,


1977,


Bharal


and


Rashid,


1979,


Mehta


and


Mohan


Ram,


1980,


Mathews


and


Rao,


1984).


Although


shoot


regeneration


was


achieved


these


studies,


rooting


and


establishment


soil


often


were


difficult


resulting


low


success


rates


for


whole


plant


development


and


survival.


Little


quantitative


and


comparative


information


on organ


yields


per


explant


type


and


the


productivity


of various


genotypes


were


reported.


This


technique


of seed


explant


culture


the


first









from


vitro-cultured


embryo


axes


and


embryonated


and


deem-


bryonated


cotyledons


that


were


whole


and


sectioned.


Optimal


quantities


of shoots


were


obtained


with


whole


embryonated


cotyledon


explants


cultured


on solidified


medium


supple-


mented


with


6-benzylaminopurine


(BA).


Excised


shoots


devel


oped


roots


vitro


upon


transfer


to medium


supplemented


with


mg l-napthaleneacetic


acid


(NAA)


Long


-term


pro-


lific


shoot


cultures


were


produced


and


maintained


using


thi


procedure,


these


and


cultures


whole


over


plants


a 34-wk


were


continually


period.


produced


procedure


from


was


suC-


cess


with


20 genotypes,


although


shoots


were


produced


quantities


ranging


from


to 14


per


responding


culture


after


10 wk.


Investigations


of factors


that


influence


the


level


of shoot


productivity


may


necessary


on an individ-


basi


genotypes.


to optimize


In vitro-produced


regeneration


plantlets


system


transferred


specific


to soil


and


placed


a greenhouse


developed


successfully,


matured,


and


seed.


No phenotypic


variants


were


observed


among


the


plants


produced


these


experiments.


Thus


system


allows


vitro


production


of morphologically


normal


plants


of peanut


at high


frequencies.


The


regeneration


response


of leaf


explants


from


the


cultivated


peanut


and


perennial


peanut,


Arachis


alabrata


Benth,


was


evaluated.


Maximum


induction


of bud


tissue


from


r 1


A m


I


I


--









with


NAA


and


BA L'-


was


previously


reported


Mroginski


et al.


(1981)


and


Pittman


et al.


(1983).


Thi


present


report


indicates


, however,


that


larger


quantities


shoots


can


be induced


with


higher


cytokinin


concentrations.


Increased


frequency


of shoot


formation


becomes


imperative


when


applying


thi


regeneration


method


to peanut


improvement


programs.


Eighty-four


percent


of the


bud


tissue


cultures


from


cultivated


peanut


continued


growth


and


development


into


shoots


within


BA L-1


and


d of


no NAA.


transfer


These


to medium


shoots


supplemented


developed


with


roots


vitro


within


30 d of transfer


to auxin-supplemented


medium.


In vitro-produced


plantlets


were


transferred


to a greenhouse


where


they


developed


successfully


Although


phenotypic


variants


were


not


observed


among


any


the


regenerated


plants,


thi


system


potentially


offers


a high


degree


variability


which


regeneration


can

was


be exploited


induced


peanut


17 of the


improvement.


20 genotypes


tested.


Genotypic


differences


organogenic


capacity


require


further


investigation


among


peanut


cultivars,


because


much


variability


was


noted


the


quantities


responding


explants.


Additional


study


of other


explant


sources


may


provide


areas


increased


organogenic


activity


use


this


tissue


culture


system.


The


perennial


peanut


leaf


explants


callused


and


pro-


m


A









following


a sequence


of development


including


a callus


stage.


This


indirect


organogenesis


pattern


primary


explant


-callus


* meristem


+ organ.


The


largest


number


organogenic


calli


occurred


on media


supplemented


with


either


or 5


produced


BA L-1


did


The


develop


majority


and


of shoot


differentiate


meristems


to become


initially


whole


plants.


As with


culture


initiation


and


optimum


shoot


meris-


temr


production,


fine-tuning


of this


tissue


culture


system


may


allow


greater


numbers


of meristems


to follow


the


normal


course


of development


germination.


This


vitro


regen-


eration


system


would


allow


the


potential


mass


production


of plants


from


improved


one


lines.


exhibiting

It also


or carrying

provides an


traits


desirable


alternative


clonal


propagation


method


crops,


such


as perennial


peanut,


that


are


not


normally


seed


propagated.


The


induction


of microspore-derived


embryos


and


the


production


of haploid


plants


from


the


cultivated


peanut


were


evaluated,


and


limited


success


was


achieved.


Microspore


survival


and


development


was


investigated


and


provided


evi


dence


that


uninucleate


microspores


respond


differently


culture.


Many


the


microspores


degenerated


within


28 d of


culture,


while


occurred


the


others


14th


remained


d of culture,


uninucleate.


with


Mitosis


approximately


uninucleate


microspores


completing


their


first


divis








Wicks,


1969),


with


additional


microspore


cell


divi


sion


resulting


complete


embryo


development.


Although


there


was


no evidence


of further


embryo


or pollen


development


from


the


binucleate


microspores


of peanut


during


the


70-d


obser-


vation


period,


additional


time


may


have


been


required


for


embryogenesis.


Also,


there


may


have


been


inhibitors


embryogenesi

microspores.

development


release

Future


from


research


of a system


anther

h should


to obtain


tissue


or degenerated


concentrate


a pure


culture


on the


these


binucleate


microspores.


Thi


will


provide


isolation


from


unresponsive

microspores


and


degenerated


to continue


microspores,


development


along


allowing

the embr


competent


yogenic


pathway.


Structures


res


embl ing


embryos


emerged


from


callus


that


was


initiated


within


the


walls


two


anthers,


but


no fur-


their


development


these


structures


occurred.


Root


organ-


ogenesis

however,


occurred

shoot de


from


somatic


velopment


did


anther


not


tissue


occur.


-derived


The


callus


formation


roots


provides


evidence


morphogenesis


and


ssible


whol


plant


regeneration,


which


would


provide


an additional


source


of genetic


diversity


this


important


crop.


The


formation


of somatic


embryos


and


callus


from


the


cotyledons


immature


zygotic


embryos


was


investigated


three


peanut


cultivars


cultured


on media


supplemented


with








require


much


research


the


future


to determine


the


influ-


ence


of genotype.


The


role


of other


factors


such


as growth


regulator


regime,


tissue


age,


and


environmental


conditions


will


also


require


further


evaluation.















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BIOGRAPHICAL


SKETCH


Alexandra


. McKently


was


born


Philadelphia,


Pennsylvania,


education.


where


She


graduate


obtained

d from U


her


pper


primary

Darby H


and


igh


secondary


School


1974


Alexandra


received


a Bachelor


of Science


degree


horticulture


in 1979


from


the


Pennsylvania


State


University,


University


Park


, Pennsylvania


She


attended


the


University


of Florida,


and


obtained


a Master


of Science


degree


agronomy


in 1981.


In 1984,


Alexandra


began


study


towards


Doctor


of Philosophy


degree


to be awarded


December


1988









certify


that


have


read


this


study


that


opinion


it conforms


presentation


and


to acceptable standards of


fully


adequate,


in scope


scholarly
d quality


a dissertation


for the degree


of Doctor


of Philosophy


Frank P.
Professor


Gardner,


Chair


of Agronomy


I
opinion


certify that
it conforms


I have


read this


study


and


to acceptable standards of


that
school


in my
early


presentation an
a dissertation


fully adequate,


for the degree


in scope and


of Doctor


quality


Philosophy


0. r -S_


Gloria A.
Associate


Moore,


Cochair


Professor


Horticultural


Science


I
opinion


certify that
it conforms


presentation and
.a dissertation f


I have


read this


to acceptable


fully


adequate


or the degree of


study


standards
a


I


in scope


Doctor


that
school


in my
early


qua] ity,


of Philosophy


Kenneth J
Professor


ote


of Agronomy


opinion


certify that
it conforms


have


read


this


study


to acceptable standards


and


that


in my


of school


presentation and


fully


adequate,


in scope


quality


a dissertation


for the degree


of Doctor


of Philosophy

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< Premn


Chourey


Associate


Professor


of Agr


otomy
4 -


certify


that


I have


read this


study


that


in my


opinion


presentation


conforms


and


to acceptable


fully


adequate,


standards
in scope


scholarly


and


quality


a dissertation


for the degree of


Doctor


of Philosophy


J /r fI


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opinion


certify that
it conforms


have


read


this


study


to acceptable standards of


that


in my


scholarly


presentation


and


fully


adequate,


in scope and


quality


a dissertation


for the degree


of Doctor


of Philosophy


Terry W.
Associate

This dissertation was submitted


the College of Agriculture


and


Lucansky
Professo


of Botany


the Graduate


Faculty


the Graduate School


was


accepted as


partial


fulfillment of


the


requirements


the degree of


December


Doctor of Philosophy.


1988


an, Co


ege


f Agriculture


Dean,


Graduate School


, as


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