Application ptant biotechRology - J-STAGE

Ishikawa Agricultural College

NII-Electronic Library Service

IshikawaAgriculturalCollege

But}. Ishikawa Agr. Coll. 26/ 15-43 (1996)

Application of ptant biotechRologyforbreeding of Ipomoea species

Motoyasu O[IANI

(haboratory of Plant Cell Breeding, Researeh Jnstitute ofAgriculturat Resourees )

Contents

Summary

Key Words

Chapter l. General Introduction

Chapter 2, Plant regeneration from calli deriyed from leaf tissue ef lvomoea species

Experiment ] . High frequency of plant regeneration from Ieafcalli of sweet potato

Experiment 2. Plant regeneration from leaf ealli of lpomoea trichocarpa El].

Chapter 3. Protop)ast culture of sweet potato

Experiment 1. Callus and root formation frem protoptasts of mesophyll and cultured cells

Chapter 4. Genetic transformation of lpomoea species

Experiment 1. Transformation of sweet potato plants by Agrobacterit"n rhizogenes

Experiment 2. Ferlile transgenic plants of lpomoea trichocarpa E]1. induced by different

strains ofAgrobacterittm rhizogenes

Chapter 5. General Discllssion

Acknowlegements

Abbreyiations

References

Summary (in Japanese)

15161618182124242727

323638383g43

Summary

ln this study, I tried to establish the necessary protocols for the application of plant biotechnology to breeding oftwo

ipomoea speeies, L batatas (sweet potato) and l, trichocarpa, The results obtained are summarized as fo11ows.

1. High frequency of plant regeneration from leafcalli ofsweet potato.

High frequency p]ant regeneration was established from ]eaf calli of sweet potato (lpomoea batatas (L.) Lam,) cv,Chugoku 25. Calli were formed from teaf segrnents on LS medium supp]emented with O.5mgtl 2,4-D, 3,OOOmgl1 yeast

extract, 5% (W/V) sucrose and O.25% (W/V) ge]]an gum. Plant regeneration occurred at the frequency of more than 25% by

transferring the calli onto the regeneration medium which was LS basal medium without any p]ant growtb regulators, The

presence of either abscisic acid (ABA) or silver nitrate (AgNO]) in the callus induction medium promoted shoot regenera-

tion. The optimum concentration was 2mgtl fbr both ABA and AgNO,, the frequency of plant regeneration being 70 and

73.3%,respectivety,

2. Plant regeneration from leafcalli oflpomoea trichocarpa Ell.

Cal]i werc induced from Ieaf pieces of lpomoea trichocatpa on the LS basal medium supplemented with O.5mgtl 2,4-D,

5mgtl ABA, 3gll yeast extract, 5% sucrose and O.2% gellan gum. These calli formed adventitious buds on the regeneration

med]a containing more than 2mgl1 BA, The highest adventitious bud formation was obtained at 1Omgll BA. Adventitiousroots were atse preduced from calli on alT kinds of the regeneration media used in the present study. There are two type of

the adventitious roots from leafcalli, thick roots (1 ,Omm in diameter) and thin roots (less than O,2mm in diameter). Whenthe carli with adventitious roots were transferred to plant growth regulator CPGR)-free LS medium, 63.3 - 90.6% ofthe roots

produced shoots, Shoots werc on]y regenerated from the thick roots. It was suggested that some adventitious shoot primor-dia were alfeady devetoped on the thick roots. Shoots rcgenerated from both leaf calli and adventitious roots could subse-

guent]y grow to planttets. Then these p]antlets were transplapted to pots containing a mixture of yermiculite and perlite. All

of them grew nonnally in the growth chamber and had norma] seed fertility. No morphological differences were obseryed

in 71 regenerated plants.




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Bulletin of [shikawa Agricultura] Co]lege No. 26 (1996)

3. Cal]us and reot formation from protoplasts of mesophyll and cu]tured celts.

Protoplasts were iso]ated from mesophyll tissue, callus and suspension ccll cultures of sweet potato. Viable protoplasts

<7,1× lO`-8,6× 1OS protoplasts/g fresh weight) were obtained from the third Io fifth ]eaves from the top of these plants,whereas younger ones, the first and the second, y3elded only smatl and fragi]e protop]asts which did not divide in the

medium. When chopped leaf tissue was incubated m sterile distii[ed water for t6 h pnor to enzymattc digcstion, the yield of

protoplasts showed 20-fold increased cempared with untreated matemal in all oflhe cu]tivars tested jn the present study. In

protoplast isolation from cultured cel], ffeshly isolated protop]asts from cultured cells were cytop[asmic-rich and 30 to

40gm in diameter, The yield of protep]ast was 1-2x1Ofi cclls/g fresh wight in both cal]us and ce]] suspension cultures, No

clear difference in the yield ofprotoplasts was observed between tsij'o cu[tivars, Abeut 13% of mesophy]1 protoplasts and

25% of cu]tured ceH protoplasts divided on the third day. When these ca[ti derived from both mesephyl] and callus cells

protoplasts wefe transferred onto the regeneration media, a few roots wcre formed from the calli p]aced on the media

containing both BA at O.5, ],O or 2.0 mgtt and IAA at O,2 mgtl, but no shoets were produced.4. [IVansformation of sweet potato plants by Agrobacterium rhizogenes.

Transgenic sweet potato plants were obtained afterAgtvbacterium rhizogenes-mediated transformation, Leaf disks of

in vitro plants were inoculated with diiferentA, rhizogenes strains, Numerous hmry roots were induced on leaf disks by both

agropine-type and mikimopine-type strains. Whole plants transformed with Ri-T-DNA were regenerated from the hairy

roots in five cultivars, These plants had wrink]ed ]eaves, altered shape of flowers, reduced apical dommance, shortened

internodes, small storage roots and abundant, frequent]y branching roots that showed redueed geotropism.

Transgenic sweet potato plants possessing both tu)tll and gus genes were a]so obtai ned from the hairy roets by infection

with A. rhizogenes containing the binary yector pBl121 an addition to the wild-type Rj-plasmid.

5. Fertile transgenic plants of ipomoea trichocarpa EII, induced by ditferent strains ofAgrobacterium rhizogenes,

Cotyledon exp]ants of lpomoea trichocarpa El], were inoculated with ten strains ofA. rhizogenes. Hairy roots were

produced from the cut surface of exp3ants by mecu]ation with a]] bacterial s[rains. No c]ear differences in rhizogenicity

were observed among the bacterial strains ofA. rhizogenes tested, Whole plants were regenerated from the hairy roots

transformed by all of the bacterial strains. These haify root-derived plants exhibited the expected transformcd phenetype,which was sexually transmitted to the progenies in Mende]tan fashion as a single dominant tocu$.

Transgenje L trichocarpa p]ants possessing bolh thc nptli and gus genes were a[so obtained frorn the hairy roots by

infection with A, rhizogenes containing the binary vecter pBI12hn addition to the wild-type Ri-plasmid,

[Key Words] biotechnology, genetic transfbrmation,lpemeea species, sweet potato, tisslle culture.

Chapter 1General Introduction

1-1. Backgrounds of this study

Conyentiona] plant breeding is mainly based on cross-breeding

and mutation breeding methods. They permit increasing genetic

variability and improving genetic composition ot'crop species, Hew-

ever, these methods have several ]imitations in plant breeding. The

cross-breeding method can not be apphed in the breeding of stcri]e

plants and i$ difficult to produce hybnd plants in interspeeific and

intcrgeneric hybridization due to the cross-incompatibility. Thus,

cross-breeding can only be applied for the species included in the

closely related taxa. On the other hand, it is genera]ly difficu]t to

control the mutation events in the induced mutauons, thus this tech-

nique has not been app]ied cthciently to plant breeding {Yamaguchi1 986). Tb overcome the limitation of these conventional p] ant breed-

ing methods, therefore, noycl approaches such as cel] and genetic

engineering techniques are now required to bc incorporated mto

plantbreeding:i.e.genetictransformationpefmitsintroducingnovel

traits to crop species and somatic hybridization technique ailows

producing the hybrids between the species in which the hybrid can

not be produced due to the cross-incompatibility,

The p]ant cel1, tissue and organ culture techniques are prerequi-

site for genetic transformatjon and sematic hybridization. Adyen-

titious shoot formation from cu]tured tissues was first observed with

roet cultures of tebaeco (Nicotiana tahacum) by White (]939).Skoog and Miller (19S7) reported Ihat organogenesis from tobacco

pith tissue cultures cou[d be regulated by plant growth regulators;

high concentrations oi' auxjn promoted rooting, whereas high lev-

els ofcytokinin stimu]ated adventitious soot formation. Since then,

promotion ef shoet regeneration by exogenous cytokinms has been

reported in a nurnber of p]ant specics. However, most of the callus

cultures of cerea]s exhibited organogenesis when they are trans-

t'crred from a medium containing 2,4-D onto a mediurn without it

or contatning IAA or NAA instead of 2,4-D (Nishi et al. 1968,

Niizekj and Oone ]97], Bajaj and Bidani 1980). In some dicotyle-

donous plant species such as celery and carrot, somatic embryo for-

mation from ca]]us culturcs was also promoted by transferring them



IshikawaAgricultural College

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M, Otani1 Application of p]ant biotechnology forbreeding of lpvmoea species

onto a medium lacking auxin (Dudits et al. 1976, A]-Abta and Co]1in

1978)). These findings suggest that regulation of shoot formatien

from cultured tissues by exogenous hormones is not simp]e,

Plant protoplasts were first isolated mechanically by cutting the

edge of plasmolysed celts with a razer (K] ercker 1 892). Ihen, Cock-

ing (1960) succeeded in isolating protoplasts enzymatical]y from

root tip ce]ls of tomato seedling by using cellulase extracted from a

cellulolytic fungus Myrothecium verrcama, Furthermore, fakebe

et al. (1968) established mass isolation rnethods of active proto-

plasts from tobacco mesophyl1 tissues by using commefcially avai]-

able enzymes, Nagataand fakebe (1971) then succeeded in regen-

eration of complete plants from enzymatically isolated tobaceo pro-toplasts. Ihese results provided the evidence that the single ce]ls of

higher plants have totipotentnature, Since then. plant protoplasts

have been used extensively in the various researeh area such as

plant physiology, biochemistry, viro]ogy and genetics, especially

somatic cell and organelle genetics. Therefore, p]ant regeneration

from protoplasts has been reperted in a number of higher p[ant spe-

cies, Success in regenerating whole plants from protoplasts even-

tually led to attempts to cembine ccl]s with different genetic back-

ground. In 1972, Carlsen et al, first succeedcd in producing the

sornatic hybrids between IVicotiana tabacum and N. tangsdbcfii by

protopiast fusion, suggesting that somatic hybridization theoreti-

eally offer possjbilities for genetic exchanges between distantly re-

lated species, However, several prob]ems have been perceived in

somatic hybridization for uti]izing in planl breeding. They were,

for example, random chromosome elimination from somatic hy-

brid eells. Iack of morphogenic capacity of hybrid cells, production

of hybrid plants with abnormal morphology and/or sterility {G]ebaand Hoffman 1980, Melchers and Labid ]974, Harms 1983, Galun

and Aviv 1983), which mainly because of genomic incompatibili-

ties as suggested by Harms (1983). Therefore, in order to over-

come these preblems, modified methods such as asymmetric so-

matic and gametosematic hybridization teehniques have been de-

ve]oped and widely examined in a large number of plant species to

introduce a part of nuelear gcnome or organe]]e genome, or te pro-

duce cytoplasmic hybrid (eybrid). Genetic transformation has been developed as an alternative

method to the somatic hybridization for the application of biotech-

nologies to plant breeding. This method is used to introduceaspe-

cific desirable gene into plant celi without concomitant transfer of

unde$irable traits, which usual]y occurs in sexua] cross and somatic

hybridization, In the 198e's, tremendous progresses were made in

the genetic transformation in higher plants {Bajaj 1989). Theseadvances have been due to the development ofefficient gene vector

systems and techniques of plant transt'ormation and regcneratien.

As a result of these pfogresses, several gcnes which relate to agri-

culturally important traits such as yirus resistance (McGarvey and

Kaper, 1993), herbicide resistance (Hinchec et al, 1993), insect re-

sistance (Barton and Miller 1993, Hilder et al, 1993) and qua]ity

improvement CSun and Larkins 1993, Rees 1995) were isolated and

introduced into plant ce]]s to produce transgenic plants without any

othcr undesirable changes. Furtherrnore, the number of fietd re-

lease of transgenic plants has been increastng since the first field

test ot' geneticalSy transformed p]ant was done in 1986, More than

one thousand (1,025) field re]ease experiments were conducted in

32 countries from 1986 to l993 (Goy and Duesing 1995), Recently

the target materials of these plant biotechnologies haye been ex-

tend to not on]y important cereals such as barley, maize, rice and

wheat, but also te horticu]tural crops. In 1994, the Calgene Inc.'s

Flavr SavrTM tomatoes, the first genetica]]y engineered plant prod-

uct were reteased into market in the United States. The tomato has

a synthetic genc that inhibits the expression of polyga] acturonase,

which norma]ly acce]erates fruit softening and contributes to the

over-npening of tomatoes. The fie]d test database of the GIBR

which is an industry associanon ot' 19 European companies involved

in biotechno[ogy, suggests that 1O to 20 other geneticalIy modified

plant products seem to qualify for market introductien before the

next century cornes CGoy and Duesing 1995),

1-2. Application of biotechnology to lpomoea genus There are two groups in lpomoea species in the seetion of

BatatasbasedonthecrosseornpatibilitywithLbatatas;oneis4t7inis

series <group A) which has cfoss ineompatibility with J. batatas

and the other is Batatas senes (group B) whjch includes, of course,

sweet potato (I. hatatas) and has the eross eompatibility with it

CTleramura ]979), Considerable traits of wild relatives belongingto group B have been used for sweet potato breeding su ¢ h as dis-

ease and insect resistance and high starch eontent, and a cultivar

Minamiyutaka which has high storage ability and resistances to b] ack

rot, root-knot nernatode arid root-]esion nematode was deve]oped

in Japan (Ono et al. ]977). On the other hand, wtld relatives which

belong to group A have not been used for sweet potato breeding

because of their cross incompatibility with sweet potato,

In sweet potato, p]ant regeneration from callus cultures has been

achieved utilizing shoot tips <Jarret et al. I984, Liu and Cantliffe

1984), reots (Yamaguchi and Nakajima 1972, Hwang et al, l983),

anthers (Sehgal 1978) and ]eaves (Sehgal 1975, Liu and Cant]iffe

1984). However, tn most cascs, thc planl regeneratlon frequency

was very ]ow and genotypic difference in regeneration ability sn]]

remains, In otherspecies ofthe genus lpomoea. a few papers were

reported on plant regcneratien frem cal]us cultures. Xue (1988)cu]tured unferti]ized ovules ofL littoralis and severa] plants were

regenerated. Liu et a],C]990) obtained regenerated plants from

stem, petio]e and ]eafexplant cu]tures of L triloba, Belarmino et

al. (]992) observed plant regeneration from adventitious roots de-

rived frorn ]eaf cu]tures of L lacunosa anct hexaploid L trijida.

Recently, Pido and Kowyarna (199S) obtained regenerated plants

from shoot tips via somatic embryogenesis in seycral diplojd strains

of L trip'da.

Plant regenefation from protoplasts od' several species in the

genus lpomoea was reported by Murata et al. (1987), Sihachakr

and Ducrcux (I987), Suga et al, (1990), Liu et al. (l99 ]), Pcreraand Ozias-Akins {1991) and Be]armino et al. (]994). In genetictransformation of ipomoea species, en]y three repores have been

published. Parakash and Varadarajan (1992) oblained transformed

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Bullctin of Ishikawa Agricu]turat Col]ege No.26{t996)

catlus and roots of swcet potato using the particle bombardment

system Neweli e{ al. (1995) obtaincd transgenic sweet potato plantsusingAginhacierium tt"nefLiciens, However, progresses ln proto-

plast cul{ure, somatic hybridization and genetic transformation in

sweet potato and its related species has been far behind these in

other tmpertant creps such as ricc and potato

Recently. some important genes for the improvevnent ef sweet

petato, such as eoat protein gene of sweet potato feathefy mettlc

virus (SPFMV) wh]ch is expected to introduce SPFMV tolerance

(Ni$htguchi and Mori 1993) and BT-toxin gencs from Bacitlusthuri.ngiensis which are expected as insect resistant genes, have suc-

cessf'uliy been isotated and characlerzzed (Vaeck et al 1987,

Fischhoff et al, 1987), Aircady, BT-toxin genes have been ]ntro-

duced into somc crop species such as eotton (Per]ak et at ]990),

ma]ze CKozic[ ct al. ]993>, potato {Chcng ct a]. ] 992). Iobacco CVieck

et al ]987) and tomato (Fischhoff et al. ]987), and expression ot'

these genes conferred insect toleranee on rhe transgenic plants and

theirprogenies.

Thereforc, tt ean be expected that biotechnologies wi11 be gradu-al]y apphcd to hreeding of swect potato and other lpomvea species

with the use of the e[1'ieient and reproduclble culture systems for

plant regeneration, protoplast cu]ture amd genetic transformation m

thescspecies,

1-3. Aim of this study and outiine of this thesis

Eslahhshment ef the efficient and repreducible culture systems

fer planT regeneration, pretoplast culture and genetic transi'orma-

uon in the genus lpomoea, as menLioned abovc, is a prcrequisrte for

breeding ot' this genus by using biotechnologies, To date, severa[

studies huve been reported on tissue and pFotoplast culture. and

geneuc transformation in the genus 1)mtnoea. 1-lowever, unlike other

important crops such as bar]ey, maize, potato anci rice, ncither so-

matic hybrid p[anLs nor transgenic p[unts with :ntroduced agricul-

turally important genes havc yet been prc}duccd in ij)omot.a spe-

aes. In this study, therefore, l examincd the pessibility of the ap-

plicatien oi' btotechno[ogies tbr improvement of the plant spccicsbelong to thc gcnus lpom"ea.

In chapler 2, et'Iicient and reproduab]e cu]ture systems tbr planl

regeneration I'rom eu]tured explants were described. In experjment

1,efl'ectoi'p]antgrowthregu1atorsandAgNO,onadventitiousshool

formalion frem [eaf exp] ant-dcrivedi ca]]us cu]tures {)i' swect p()tato

was exammed. In expenment 2, eft'ect of 6-benzyladenine (BA)

andi otherconditions t'or adventitious shoot fermatic)n from adven-

titious reo{s derived 1'rom Jeai ca]1i of l. trtchocaJ7pa were exam-

med to estahksh an ei'fi cient p]anL regeneration systern.

Inchapter3,cfilcicntmethodst'orprotoplastisolat]onandcolony

formation from both mesophyll tissues and cu]tured ee]ls were de-

scribcd Roet different]ation from the protoplast-denved call] was

ajsodcscmbcd.

In ehapter 4, genetic transformation of' sweet potato and J.

tri(/ho(/aipa usingAgrohacteri"m rhtzogenes was desenbed, ln ex-

perimen[ 1, effieient production ot' transgenic sweet potato p]antsby A t'hi.7ogenes-mediaLed transformation is described. The pro-

duct]on of transgcnic sweet potato p}ants harbonng both nptll and

gtts gene by us]ng a bmary vector system based on A. rhizogenes-

Ri ptasmid was a]so descmbed in this expenment. In experiment 2,

producUon of ferti]e transgenic plants ofL tric/hoc/arpa induced by

A, rhizogenes was. described. [n this experiment, transgenic plantsof L tri(tho(tarpa were ei'fieiently produced using different strains

ofA. rhizogenes through hairy roet formation and plant regenera-

"on and the inhcritance of the transformcd traits to the progenieswas examincd. Transgenic ptants ot' L tricho(/arpa possessing both

nptii and gus gene were a]so produced by usmg a binary vector

system based on A, tVnzogenes-Ri p]asmid and the genetic analysis

of these introduced traits was conducted in this experiment.

Finally, in chapter S, the general signifi cance ot' the results ob-

tai ned in this thesis was summarized and discussed. Fllrthermorc,

importance of biotcehnology ]n p[ant breedmg of importan[ crops

includ]ng lpomoea speeics -'as discussed.

Capter 2Plant regeneration from calii derived from geaf tissues of lpomoea species

Experiment 1 : High frequency piant regeneration frem

Seaf calli in sweet potato

Iemtroduction

Ptant regeneration f'rom ca[li and tissue cultures js an important

step for genctlc manlpulatlon ln s-,eet potato. Slnce sweet potato

leavcs havc a goed suscepnbdity to infeetion with various wt]d

strams ol'Agrvbac/teruun rhii.ogene.s (Experiment l, Chapter4), ]eaf

Lissuc is alse expected to be a suitah]e matenaH'or infcction with

Agtoha('terit.un tttmctLi('tens. Although p]ant regeneration from ]eaf

tissue-demved callus eu[tures of sweet potato has already been re-

perted (Sehgal 1975, Liu and Cantlit'fe 1984), low plant regenera-

ti{)nfreguencicsobtainedinthesestudiesrnayhampcrthetransgenic

p]ant t'ormation by uti]izing disarmed Ag")hacterium tuniojZiciens

as a vector.

In [his expenmenl, 1 descmbe the effieient plant rcgeneration

method from leafcalh oi' sweet potato cv. Chugeku 25.

Materials and methods

Plantmaterials

Chugoku 25. a c"]L]varof Lt?omoea batatas (L.) Lam. was used,

To ebtam Tn vitro plants of sweet potato, meristcm culturc was per-

formed accordmg to the method described in expenment ], Chap-

ter 3 ln vitro plants were grown on LS medium containing O.25%

(W/V) gcllan gum and lnckmg plant grewth regulators under 4,800

lux fiuorescent Iighl t'{}r 16 h at 26'C.

I.eaf' ca]lus inductien and sheot regeneration

'Fhc

apical third to fiflh fully expanded ]eaves ofin vitro plants

were cut ]nto rectangular pieees about 1O mm ]ong. Thcsc leaf

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M. Otani 1App]icatien of plant biotechnology for breeding ot' lpomoen species

pieces were inoculated for callus induction onto LS rnedium supple-

mented with O,S mgA 2.4-D, 3,OOO mg/1 yeast extract, 5% CWIV)sucrose andO,25 % gellan gum. In someexperiments, various con-

centrations of ABA (O, O.5, 2 and 5mgtl) and AgNO, (O, O.2, 5 and1O mgfl) were added in this ca]lus induction mediurn andtor shoet

regeneration medium to examine their effect on shoot regeneration,

For shoot regeneration, ca]li formed t'rom leaf explants were trans-

ferred onte LS medium supplemented with various eoncentrations

(O, 2, 5 and 1O mgt1) of BA, 3% (WIV) sucrose and O,25% (WtV)

gellan gum, Plant growth regulators and AgNO, wcrc added to the

media prior to autoclaving and the pH of the Tnedia was acijusted to

5.9. The cultures were incubated at 26bC under 4,800 ]ux fiuores-

cent light for 16h,

lhe adventitious shoots excised frorn }eaf ca]li grew and roeted

well after transfer onto O.8% agar (W/V)-solidified LS medium

without any growth regul ators. When the shoots of these p]antlets

beeame 1OO to 150 mm long, they were transferred to pots contain-

ing a 2:1 (V/V) ratio oL vermiculite and perlite rnixture and placed

in a centainer covered with transparent piastic film to maintain a

high re]ative humidity. They were watered twicc a week and main-

tained at 26℃ under a 16-h photeperiod with fluoreseent lamps

(14,ooO lux) in a growth chamber for 30 days,

Tab]e 2. Effect of ABA en shoot regeneration from leaf callus of

sweet potato cv. Chugoku 25

ABA concentration

of callus ind-ction

medium*L

No uf cal]i

transferred*2

No. of calliformlng shoots

(mg/1}o0525

505050so

(%)13{26,O)24

{48.0)35 C70.0)31

{62.0)

*L Rcgcneration medium , LS med]um supplemented with 30 gn sacrose

and 2 5 gA gellan gurn

*i Leaf cal1] were produced on LS med"]m suppleTnented vvith O.S mgA

2.4-D, 3,OOO m!n yeast exlract, SO gll sucrose and 2.5 gn gellan gum.

Table 3, Effect of AgNOi en shoot regeneration from ]eaf catlus

of sweet potato cv, Chugoku 25

Concentrat]onofAgNOynthemedium

callus :nduction

medium*iregeneratlon medium'?

No, of ca]h

transferred

No. of calli

forrning shoots

Results and Discussion

(mgtboooo (mgtbo2510

53312930

{%)14(26.4}S(16.1>7

(24.1)4(13,3>

Effect of BA on shoot regeneration from leaf calli

Friable calli pale ye]]ow in co]or were initiated sij'tthin 7 days

from a]most all ofthe leaf explants en LS medium contaimng O,5

mgtl 2,4-D and 3,OOO mgll yeast extract,

Shoot regeneration from eallus cu]tures is genera[ly induced bythe addition of cytokinin to the culture medium (Fhck et a]. 1983),

In lpomoea trichocarpa, a wild relatiye of sweel potato, our previ-

ous study a]se showed that shoot regeneration from lcaf-calli was

only induced en the media containing rnore than 2 mg!1 BA, and

that 1O mgll was the most effective (Experiment 2, this Chapter>.

In the present experiment on effect of BA, hewever, the highest

frequency (46.7%) of shoot regenerat[on from ]eaf-ca]li was ob-

tained in the medium without BA and addition of BA to the regen-

eration medium acted inhibitorily with the increase of its concen-

2510 eoo 302525 22 (73.3}

6 (24.0)

7{28.0)

2510 2510 32S433 2I {65.6) 8<14.8)

6(182)

Tab]e 1. Effect of BA en shoot regeneration frem teaf ca]]us of

sweet potato cv. Chugoku 2S

*i Cal]us mduction med:um

' LS medium suppleme(ited with O.5 mg/[

2 4-D, 3,OOO mgll ycast extraet, 50 gt] sucrose and 2 5 gll gellan gum.

*! P]ant fegenfation medium . LS mediurn supp]emeated with 30 g/1

sucrose and 2,5 gll gellan gutn,

BA eoncentratien No of calli

of regeneration transferred'!

medium*[

No of cal]ift)rmuig shoots

tfation CI'able ], Fig. 1a). Carswell and Locy (1984) also observed

simi]ar resu]t in whieh highcst frequency of shoot formation from

cultured leaftissueg was obtained in the medium without BA, These

findings suggest that [eaf ca]li of sweet potato eontained enough

amount of endogenous cytokinin and that exogenous supp]y of a

supra-optimum eonccntration of cytokinin inhibited the shoot re-

generatlon,

(mgh)o2510

30502324

<%)14

(46.7)I7(34.0)8

(34.S)3(12.S)

s] Regeneration medium / LS nledium supplemented with 30 gt1 suerose

and 2.5 gl] gellan gum.*i

Leaf calli were produced on LS medium supplemented with C) 5 mgll

2 4-D, 3.000 mgn yeast extract, 50 gfl sucrese and 2 5 g/1 gel]an gum

Effect of ABA on shoot regeneration from leaf cal]i

Caltrformed from leat' explanls on ca]Ius inductien rnedia con-

taining various concentrations ot' ABA (O, O,5, 2 and 5 mg/t) were

transferrcd onto the shoot regeneration medium which was LS me-

dium containing no p]ant growth regulators. Neithcr callus induc-

tion ner growth were at'feeted by the addition ofABA to the callus

induct]on medium. Howeyer. the addition ofABA was effective

for shool regenerat]on e$pecia]]y at 2 - S mgft (lhbte 2). Jn both J.

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20

Bulletin of Ishikawa Agricultural Col]ege No.26 clY96)

'tw,:'G t

tt.t

Fig] a-c P] ant regeneration from ]caf tissue-denved ca]lus cuttures ofsweet

potatocv Chugoku 25

a Advei]titieus shoot regeneratien from leaftissue-denved ealI-s

cu]tures Bar = 20 mm

b Adwarf mutant regenerated from leaf callus (;rght) and control

ptant {left} grovL'ing ln pots

c Tuberous roots o[' a dwarf mutant Tegenerated from teaf callus

{ieft} and centro] plant cnght)

trichocarpa and L trijida, we also obtained similar results in which

shoot fer[nation from the calli was achieved only when the callus

inductton medium conta]ning 5 mg/1 ABA was used (data not

shown). Thorpe (1978) has suggested Ihe lnvolvement ofgibberet-

lin in bud formation in tobaceo. The inhibition by exogenous g]b-

befe]]in m)ght be because the ussue synthesized the hormone in

quantities optimal for the organogenie process, In sweet petato the

promotion ef shoot formation by the addition ot' ABA to the cal}us

inductien medium may be explained en the basis of supraoptima]

levcl of endogenous gibbere]]in in cultured leaf exp]ants. There-

fere, it seemed that the presence of AB,AL in the ca]]us induction

mediurn might be an important ractor for promoting shoot regen-

cratien frem ]eaf ca]h in the lpomoea genus.

Effect of AgNO, on shoot regeneration from leaf calli

Callus induction and callus grewth were not affected by the

addition of AgNO"o thc eaMus ]nduct{on mcdium, By contrast,

the presence of 2 mgt] AgNO, in thc callus induction mcdium was

effective for promoting shoot regeneration from the ca]]i (Tlable 3),

However, higher coneentrations ot' AgNOJmore than S mgll) in

the callus induetion medium had no er rather an inhibitory effect en

the regeneration. The beneficial effect ofAgNO. has been reported

in pollen embrye fonnation I'rom anther cu[Lures of Brussels sprouts

(Biddington et al, 198g, Ockendon and Mclenaghan 1993) and tel-

rap]eid wheat (Ghaem] et a]. 1994), shoot regeneration fFom cailus

culturcs of wheat, Ni(totiana pl"mhagtntljolia (Purunhauser et a].

1987) and maizc (Songstad et al, 1988). and direct shoot regenera-

tion from coty]edonary explan{s of Chmese cabbage (Chi and Pua

1989) and sunflewer (Chraibi et al, l99D, In those ptant speeies,

the stimulation of morphegcncsis was occurred by the addition of

1,3 to 17 mgl] AgNO,. AgNO, is knewn to be a potent inhibitorof

ethylene aetion in p]ants (Beyer l976). Therefore, endogenous eth-

ylene of the leafexplants of sweet petalo may have acted inhibitori1y

to the induction of calli with good regenerat]on ab]lity, which might

be overcome by AgNOi ]n the caitus induction medium.

Characterization of regeneration plants

AII of regenerated plants survived with this aee]imatization pro-

cedure. Among 100 regenerated planls eb{ained, onty one p]ant

was morphologieal]y abnorrnal. This abnormal plant showed dwarft

ness (F]g. Ib) and failed to preduce tubereus roots (Fig. Ic). De-

spite this abnormal plant, thc frequency ofthe somac]onal variation

seems te be re]atively ]ew Therefore, the plant regeneration sys-

tem established in this study could be efficiently used for the ge-

netic transl'ormakon sludies,

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M. Otani 1Application of plant biotechnology for breeding of lpomoea species

Experiment 2 : Plant regeneration from leaf ca]li of

lpomoea trichocarpa Ell.

Introduction

Table 4. Formation of adventitious buds and roots from leaf

explants-derived calli which formed on the medium

containing ABA on the regeneration media.

Regenerationmediurn*

L trichocarpa is a wild relative belonging to 4fi7nis series (groupA) which has cress incompatibility with sweet potato and distrib-

utes in the Central America area. It has the biggest flower size

among 2X species involved in lpomoea species, and forms the tu-

berous roots. It also blooms numerous fiowers and is expected as a

new ornamental crep,

Establishrncnt of the methods for plant regcneration from cul-

tured tissues might provide the basis for pessible cell selection, so-

matic hybridization and genetic manipulation. In the wild relatives

ofsweet potato, there have been a few studies related to tissue or

organ culture. Kobayashi (1984) cuitured ]eaf and stem tissues of

30 strains of L trijidu (2X, 4X and 6X) and found that only one

strain (8043, 2X) showed shoot regeneration from the cal]i. Sugaand Irikura (1988) also reported p[ant regeneratron from leaf tis-

sue-denvcd ca]lus ofa strain efL trijida (K221). In the species of

group A. Liu et al. C1990) reported the plant regeneration in stem,

petiole and leaf explant cultures ofL triloba L., and Betarmino et

at, (1992) obtained regenerated plants from ]eaf cuttures of L

lacunosa L.. Hewever, there ha$ been no report on the cell and

tissue cultures of L trichocar7Ja. In the present study, I describe the

conditions necessary for plant regeneration from leaf exp]ant-de-

nved calli of L trichocarpa,

IAA BANo,ofcal]i

No,ofcalli Ne.ofcalli

transferred withbuds wlthroots

(mgh}oOO.2O.5oO,2O.5oO.2O.5oo(mgtl}oO.5O,5O.52224446103331323I3232293231293t35

(%)o(o)o(o)o(o>oco)2(63)3C

9.4)2(

6.9)3(

9.4)2(

6.5)5U7.2)5(14.7)7(20

)

{%}33{lee }30{

96.8}23{

87.5}31{1oo

>32{1oo

}30(

93.8}29{too

)31( 96.9}31(1oo

}29(100 )34(1oo )35(1oo

)


* LS basal mediurn contemning O.2 % getlan gum and 3 % sucrose

Table 5. 0rganogenesis in ]eaf explants-derived calli which

produced on the callus induction medium without

ABA.

Regeneration medium'

IAA BANo

ofca]lt No ofcalli No.ofcalli

transferred with buds with roots

Plant materials

Plants of ipomoea trichocarpa Ell, which were derived from

the seeds collected in the Florida, United States ef America was

used in this study. Stem segments (ca, 1.5 cm long),each includinga terminal bud, were excised from L trichocarpa plants that had

been grown in a growth chamber, and sterilized in 3% {vlv} sodium

hypochlorite solution for 4 and half minutes, rinsed three ti mes with

sterile distatled water, and then placed on Linsmaier and Skoog CLS)medium (1965) supplemented with O.2% (wlv) gellan gum, Cul-tures were kept at 26dC with a 12-h photoperiod (6,ooO lux). Jnvitro-grown plantlets thus estab]ished were used for callus induc-

tlon.

{mgh)oO.2oo(rng/l)o2410

20232633 oooo

<%)20(IOO

)23(1oo )

24 ( 92.3)

31( 93.9)

Callus induction and shoot regeneration

Calli were induced from leaf explants derived from 30 day-old

aseptically grown plants. Fully expanded leaves were cut into rect-

angular pieces about 1O mm long and inoculated for callus induc-

tion onto LS medium supplemented with O.5 mgtl 2,4-dichlorophe-

noxyacetic acid <2,4-D), 3,Ooo mgA yeast extract, 5% (wtv) sucroseand O,2 % (wlv) ge}Ian gum. In some experiments, 5 mgtl abscisic

acid (ABA) was added in this callus induction medium to examine

its effect on shoot regeneration. For shoot regeneration, calli formed

from leaf explants were transferred onto LS medium supplernented

* LS basal medium contarning O.2 % gellan gum and3 % sucrose.

with various concentrations of BA (O, 2, 5 and 1 O mgt]) and IAA (O,O,2 and O.5 mg/I), 3% (WIV) sucrose and O.2% (W/V) geltan gum,Plant growth regu]ators (PGR) were added te the media prior to

autoclaying and the pH of the media was adjusted te 5,9. The cul-

tures were incubated at 260C under4,8oo lux fluorescent light for

16h,

The adventitious roots derived from leaf segment calli were

transferred for shoot regeneration onto O,2% gellan gum (WIV)-solidified LS rnedium without any PGR,

The adventitious shoots deriyed from both calli and adventi-

tious roots grew and rooted well after transfer onto O,2% gellan

gum (WIV)-solidified LS medium without any PGR. When the

shoots of these plantlets became 1OO to 150 mm long, they were

transferred to pots containing a 2/1 (YtV) ratio of vermiculite and

perlite mixture and placed in a container covered with transparent

plastie film to maintain a high relative humidity, They were wa-

tered twice a week and maintained at 26eC under a 16-h photope-riod with fluorescent ]amps (14,OOO tux) m a growth chamber for

30 days.

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Bulteun ot' ishikawa Agncultural Cotlegc Ne.26{1996)

Table 6. Fofmation of thick roots on regeneration media.

Regeneranon medium* No ofcalH

transferred

No of' calh

with thick roots

Tablc 7. Shoot fbrmauon frem advcntasous roots on LS hormonc-frec

medium,

iAA BA Root fo"ning ined]um'

(mgll)ooCL2U.5o{].2O.5o02O,5eo(mgl1)eO.5O.5.05.222444b10 (%)33 S(15.2)

3. 1 13 (4 L9}

32 7 (21 9}

3t l2 c38.7)

32 g(25}

3. 2 9 (2 81 . D29 5(17./)

32 14(.43.S)

3t 61f94)

29 9C31)

31 4{H81)

35 6{17 1)

IAA

No oi' roots

BA transfeffedNo

ofroots No ofshoots

withshoots t'ormed

{mgll)o0205o02O.5oO,205(mgtl)e.sO.5O.5222444

11283t323529323029

(%･)24

(77 4)23

(82.1)27 {87.l)2I

(65 6)3. e (g5.7)24

(g2.8)29

c90.6)l9

C633)24

c82.8)

(%)72

C232.3>

sg aozl)97(313 >67 C209.4)85 C242.9)

84(2g9.7)

125. {390.6)

7t{236.7)

S7(300 >

i LS basahnedium contammg O2 % gellan gum and 3 % sucrese

* LS basa] medium containTng O2 % gellan gutn andi 3 % sucrosc

Results

Callus formation

Friabte calli with yel]ow to yellow-green coaor werc initiated

with]n 5 days from almost all of !he leaf explants on l.S mcdium

contain]ng O.S mgll 2,4-D, 3,OOO mgl] yeast cxtraet (Fig. 2a). Nei-

ther ca]lus induction nor growLh were affected by the addibon ot'

ABA to the ca]lus mduction mcdium

Effect of ABA on shoet regeneration from leaf cal]i

Cal]i fonned frorn leafcxplants on caltus inducnon ]nedia with

or without Srngil ,ALBA were transferred onto the shoet rcgeneration

mediuni whtch was LS medium containing. varioui ceneentration

ot' BA and IAA, The addiL]on ot' ABA to lhe calius inducuon me-

dium was ef±'eetive

for shoot rcgeneration On]y the calli which

formed on the media conta]ning ABA preduced shools CTablc 4),

whi[e no sheot regcncrat[on was observed in the ca]li produced on

the media without ABA (Ttlble 5)

Eft'ect of IAA and BA on shoot regeneration frorn leaf calli

Shoot regeneration was obsefved from the calli formed on the

media contaming 5 mgt] ABA within 12 days after transfer ento

regcnefation media (Fig. 2b). Supplementatiun of IAA to the re-

generatien medium had no clcar ef['ect on shoet regencration {Table

4). Shoot regcneranon d]d nol occur on the ]nedia with or without

low concentrations {)f BA (O.S mgll), while the cal[i on the media

containing more lhan 2 rngl1 BA p;oduced adventitious sheots, The

most eft'ect]ve concentration ef BA was 1O mgA at -･hich the ghoot

regeneration frequency reached 20q,

Shout regeneration from adventitious reots

Advent]tious roots werc formed from the ieaf segmemt-ca]h with

high freguenaes <87.5 - 1OO%) on al] k]ndsot' the regeneraLion mc-

dia used in thts study (Tabte 4), Thcrc were two Lypes of the rooTs

formed t'rern the calh, one was thick roots " mm in diameLer) and

the othcr was thin roots (O.2 mm in diameter) (Table 6, Fig, 2c),

Table 6 shows Lhe freqlleneies of thick roots formation from ]eaf

$egment-ca-i en the regenerat]on rnedia after 50 days of culture.

'Fhe

thick roots wcrc produced from the calli at a t'requency of 1 1,8

- 43,8%. The mosr etTcctivc mcdium for th]ck root forrnatien was

LS mcdium supplementcd with 4 mgt] BA. When the cal]i w]th the

thick roots were Lransrerred onto plant growth regulator-free LS

medium, 63.3 - 90,6% of the reots produccd adventitious shoots,

while rTe shoer was regenerated frorn the thin roots (fable 7, Fig.

2d).

Characterization ef regenerated plants

All thc shoots denved from both calli and advcntitious roets

could subsequenLjy grow to plantlets when Ihcy wcre transplanted

onto O,2%･ Cwlv) ge]lan gum-so[id)l'ied LS medium without any

growth regulators Al] oi' lhe regenerated plants survived with the

acclimatv.auon procedure. Arnong 71 regenerated plants obtained,

ne morphoio.aieal abnormality was obscrvcd CFig. 3). These ptants

grcw to maturjt.y, set seeds and had the same mofphology as those

deni,ed i'roin seeds.

Dis¢ ussion

In the present study. supple]nentation of ABA at 5 mg/1 to the

caNus induction medium ",us efi'ective I'er shoet regeT}cration of L

irtckocarpa. Simtiar etTcct ofABA was atho observed in sweet

potale {Chapter 2, Experm}ent b. Therefore, it seemed that Ihe

presence ol'ABA in the callus intiuckon medium migh{ be an im-

portanl i'actor for promoting shoot regeneration t'rom leaf calk in

the ij)vtnoea genus.

The present study shewed that h]gh concentration of BA gave

high t'requcneyofshoot regeneration. In sweet potato cv. Chugoku

2.hl, the presence of BA did nc)t promote shoot formation from leaf

segmcnt-denved calh (Chapter 2, Expenment -. 11iese resu]ts sug-

gested that endogenus cytokinin of L trtchoca rpa seemed to be not

tso high eneLLgh as that ot s-'eet potato for inducing shoets.

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M.Otani 1Application of plant biotechnotogy for breeding of ipomoeti species

Fig

Fig 3.A regenerated plant ef l

chambef,

trichocarpainapotgrowinginthegrowth

There were twe ways of sheot fegeneration from Ieafexptant-

derived calk, one was the direct formation of adyentitious shoots

from the cat]i and the other was the shoot regeneration from thick

adventitious reots formcd from the calh on the regeneration me-

dium. The highest frequency of dircet shoot regeneration was 20%,

whereas the frequcncy of shoot regeneration from the thick roots

reached to 90,6%. Be]armino et al. (1992) ebserved the formation

of both root types and no dircct shoot regencration from the leaf

ca]1i of i. Iacunosa and L tr(f}'tin, while shc}ots were only producedfrom the thick roots and the t'requency ofthick roots regenerating

shoots were 74.1% in L trofl'da and 16% in L tacunosa. Similar

fesults were also obtained in L trttuha (Liu et a]. 1990) and sweet

potato (Carswcll and Leey 1984, Belarmino et ai. ]992). ]t is sug-

gested that in sweet potato and its wild relatives, the frequencies of

d]rect shoot r'egencrat]on frorn cal1i were re]atiyely low, while ad-

ventttious roet formation occuged at high freguency in genera] and

shoot was regenerated from the adventitious roets at high frequency,

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24

BuHetin of Ishikawa Agncultural College No, 26 (1996)

These findings suggest Ihat the thick reot forrnation from the callus

cu]tures is an important step to inducc p]ant regeneration in wild

relatives of swcet potato and that this shoot regeneration method

may providc a new approaeh to overcome the low ability for p}ant

regeneratien from the cultures in re ¢ a]citrant lpomoea spccies.

Smce no merphologica] abnorrna]ity was observed atneng 7l

regenerated plants obtained, it seemed that no somaclonal vanation

occurred in the plant regeneration process from ]eaf segrnents.

Therefere, the planu'egeneration system established in this study

could be efficiently used for the genet]c transformation stuches,

Chapter 3Protop}ast cunture of sweet potato

Experiment 1 : Callus and root formation from

protopAasts of mesophyil and cuitured cells

Introduction

Protop}asts offer many potentia[ utikzauons t'or ptant b[eeding

through sornatic cel] hybridization and direct gene transfer. An ef-

ficient system for pretoplast isolation and culture is important to

reatizetheseporentials.

In sweet potate, somat]c hybridii.at]on techn]gue is expected as

thc means to introduce nevet traits from wi ld re]atives and to obta]n

intercultivar somatic hybnds, which are dMleult to pruduce by con-

ventional crossing ]nethod, A[though plant regct]eratien t'rom pro-

top]asl has aiready been repetted <Murata et al. 19g7, Shthachakr

and Ducrcux 1987, Perera and Ozias-Akins 1991, Belarmino et al.

1994), the plating efficiencies of these sludies were relauvcly low,

[t ]s generaliy considered that mesophy]l ttssue is a suTtabie

source of protopiasts, since ]arge amount ef tlssues fof protop[ust

]sotation can bc ebtamed l'rom leat' b]ades. However. isolauon of

mesephyl] pretoplasts o[' sweet potato has not been reported be-

fore. Altliough Bidney and Shepard (1980) succeeded m isolation

petiole pretoplasts or sweet potato and ferming callus 1'fem thcm,

Lhey did not succeed in iselatmg mesophyl] proteplasts. One of the

main reason for the d]fficulty in proteplast culture of s"'eet potato

may be the use of protop]asts lso[ated fronunLuct planL organs.

Shepard and r[k)ttcn

{i977) and Shepard et al, {]980) have preposed

that the potato <Solanum tuberosum) plants to be uscd for preto-

p]ast isolation shou[d be grown under prcc]sely controlled condi-

eons ot' nutrient, temperature, hghl intensity. and pholoperied, oth-

erwise the protoplasts might fail to undergo division regardless ol'

the eulture medium. Schenek and Hoffmann Ci979) alse reportcd

simiian'esu[ts in Biusstca campestrts and B. ote racea In the present

study, I descmbe the successfu] results on the ca]lus fortnation t'rom

pretoplasts and thc regeneration of roets from the calh of sweet

potato using in vitro grewn planllets tts the source of mesophy]1

protop]asts.

For the succcssfu] protep[ast culture, cul{ured cells such as cell

suspension cultures and callus cu]tures may have advantage against

to protep}ast release from intaet p]ant organs. As euitured eells are

already conditioned to growth in v]tro, they may re]ease a number

of culturable protoplasts. In the present study, therefore, l also de-

scribe the cal]us formation and establishment of cell suspension

culture in sweet potato. Isolation and culture of protoplasts from

cu]tured ce]ls ot' $weet potate is a]so described.


Plantmaterials

Five cu]tivars of lpomoea batatas L,, Beniazuma, Benihayato,Chugoku 25, Koke] l4 and Tbsabeni, were used, Meristcm cuLturc

of these cultivars was performed to obtain in vitro plants. fo obtain

the fresh shoots as the souree of meristems, the potato tubers were

incubated in a growth chamber under 16h-photoperiod with 4,800

]ux fiuorescent ]]ght at 260C. The excised [ips ef sprouting shoots

were ster]]tzed in 2% (vlv) sodium hypochlonte solutton for5 min.

rinsed three times with sterile water, and were placed on LS me-

dium (l96S) supp]emented with O.2 mgMAA and 2 mgA BA, Plant-

lets were kept on PGR-free LS med]um. These in vitro plants were

used forcatlus mduction and mesophytl protoplast ]solation.

Callus induction and establishment of suspension cultures

Root tubers of Koket 14. and petJole and stem explants of both

o[' Bemtazuma and Kokei l4 were excised frem in v]tro plants and

inocu]atcd onto thc cat[us induction medium which was LS me-

dium supplemented "'ith O.5 mgll 2,4-D, 5 mgll ABA, 3,OOO rngt1

yeast extract (Nakarai tcsgue, lnc.). 5% <WIV'} sucrose and O.2%

{WIV) gel[an gu]n. Cuttures were incubated at 26DC with continu-

ous i]lurninanons. The light -'as providcd with 11uorescent tubes

{Toshiba FL40S-D, 38ymol m'!･s'r). After 20 te 30 days ofculture,

thc calli t'ormcd were transferred mto ltquid caltus inducnon media

{25 mb tn 50 m[ Erlenmeyer tlasks. The fiasks were placed on a

gyratory shaker at 1OOrpm and mcubated under the same condi-

tions. The celts were subcultured every 7 days by removmg half

(12.5 n]1) of thc ]iquid mcdium with cc]]s, and adding the same

volumc of frcsh medium, The suspensien cu[tures maintamed ibr

one year were used for the study.

Protoplastisolation

Mesoph.i,tlprotvptast.

Thc apica[ third to fifth ful]y expanded [eaves ofthe p[ants ]n

vitro wcrc excised and chopped with a surgTcat knife. The pieces of

]eaves we[e soaked in stenle disv11ed water for ]6 h {over night) in

the dark ut 260Cl. Al'ter this treatment, the ]eaf pieces (about 200

mg) werc p]aced in a plastic Petri dish (60× 15 mni) containing 5 mt

of an enzyme solution at pH S.6 conta]ning 2% (w/v) Cellulase

Onoiuka RS, O 05% (wlv) Maeerozyme R-10, O.3% <w/v}Pectolyasc Y-23, O.5% (w/v) hemice]]ulase, ]% (wlv) Driselase,e 5M sucrose and MES, Thc dishcs werc shaken al 7S rev,lmin for

4-5 h m the dark at 26eC, and the protoplasts were then fi]tered

through a 30um nylon mesh and centnfuged at ]OOxg for 9 min.

Floating protoplagts were collccted and washcd with W5 solution

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M. OtaniI Application of plant biotechnology foT breeding of lpomoea speeies

(Menczel and Wblfe 1984) three times for purification. The yield

of protoplasts was determined by counting with a haemocytometen

Protoplastofculturedcell:

Twotypesofculturedcellswereexaminedforprotoplastisola-

tion; one was 14-day old callus freshly formed from inoculated ex-

plant on the callus induction medium, and the other was Ihree to

four-day old cell suspension ¢ ultures, maintained for one year un-

der the conditions described above. Tbn ml of suspension cu]tures

were filtered through a 30pm nylon mesh and cultured cells were

trapped on nylon mesh surface. About 5oo mg of cal]i or cel [s from

suspension cultures were suspended in 5 ml of the same enzyme

solution as used for mesophyll protoplast isolation. The enzymetreatment of cultured cells and purification of isolated protoplasts

were perfbrmed by the same procedure as used for mesophy]1 pro-

toplastisolation.

Protop}astsculture

Protoplasts of both mesophyll tissue and cultured cells were

cultured by the same method. The purified mesophyl] protoplasts

ofcv. Beniazuma and cultured cell pretoplasts of evs. Beniazuma

and Kokei 14 were resuspended in the liquid cu]ture medium to

giveaconcentrationofO,4-1.0xlptprotoplastslmlinparafilm-sealed

plastic dishes (60× 15 mm). The culture medium was modified N6

(Chu 1986) which contained (in mgA): (NH,),SO, (463), KNO,

(2830), KH,PO, (400), MgSO,･7H,O (185), CaCl,･2H,O (166),

MnSO,･4H,O (4.4), ZnSO,･7H,O (1 ,5), H,BO, {1.6), KI (O.8), Fe-

EDT;dL{42),glycine(2.0),thiamine-HCI"O),pyridoxine-HCI<O.5),

nicotinic acid (O,5), inositol (1OO), 2,4-D CO.1), kinetin (O,5), O, 1M

sucrose and O.3M mannitol, After 14 days of culture, 2 ml of cal-

lus-forming medium, which was LS basal medium supplemented

with O.5 mgt1 of 2,4-D, 5 mgA ofABA, 3,OOO mgfl of yeast extracts

and O,4M sucrose (final concentration), was added. Small calli

developed in the liquid medium were plated onto O,2% (WfV) getlangum-solidMed callus- forming medium. Four weeks after the p]at-ing, the calli were transferred onto regeneration medium supple-

mented with BA (O.S, 1.0 and 2.0 mgA) and IAA {O.2, O,5 and 1.0

mgA),

Results and discussion

Protoplastisolation

The plants of sweet potato in vitro proved to be a good source

of leaves for isolation ofmesophyll protoplasts. Viable protoplasts

(7,1× 1O"-8.6× 1OS protoplaststg fresh weighO were obtained from

the third to fifth leaves from the top ofthese plants (Fig. 4b), whereas

younger ones, the first and the second, yielded only small and frag-

ile protoplasts which did not dMde in the medium used in this study.

When chopped leaf tissue was incubated in sterile distilled wa-

ter for 16 h prior to enzymatic digestion, the yield of protoplasts

showed 20-fold increase (1.5× 106-1,2× lO' cells/ g fresh weighO

compared with untreated material (7.1×104-8.6×1OS cellsf g fresh

weight> (1labie 8) in all of the cultivars, especially cv. Benihayato,

cv, Chugoku 25 and cv. [[bsabeni. The effectiveness ofwater treat-

ment has previously been reported by Butt (1985) who achieved a

high yield of viable Ieaf mesophyll protoplasts from some mature

deciduous woody-plant species by washing chopped tissue for 30

minutes prior to enzyme addition.

In protoplast isolation from cultured cells, freshly isolated pro-

topiasts from cultured cells were cytoplasmic-rieh and 30 to 40pm

in diameter (Fig. 4c). The yield ofprotoplast was 1-2× 1Ofi cells/g

fresh wight in both callus and cell suspension cultures. No clear

difference in the yield of protoplasts was observed between two

cultivar$.

Protoplastculture

Callus formation has previously been reported only for petiole

protoptasts of sweet potato by means ef ce]1 layer- reservoir system

(Bidney and Shepard 1980). In the present experiments, both me-

sophyll and cultured celt protopiasts were able to develop into calli

rapidly in a simple medium. The first divisions in mesophyil and

cultured cell protoplasts occurred 2-3 days after the start of culture

in the modified liquid N6 medium (Fig. 4d). About 13% of meso-

phyll protoplasts and 25% of cultured cell protoplasts divided on

the third day, They continued to divide and formed cell colonies

(O.Smm in diameter) after 1 month of culture (Fig, 4e). The cel]s

grew rapid]y after the cal]us- forming liquid mediurn was added and

formed small calli (2mm in diameter) 50 days after adding the me-

dium {Fig.40. The small calli were then transferred to the gellan

gum-solidified callus-forming rnedium, and grew to about 5 mm in

diameter within 2 weeks. When the calli derived from both meso-

Callus formation and establishment of cetl suspension cultures

Callus formation from all types of explants started within 1O

days and all of the explants of two cultivars produced calli after 30

days of culture, There were no differences in eal]us production

among the different explants, The calli were fmab]c and white in

color, Tsai and Lin (1973) reported that 2,4-D was the most effec-

tive auxin in both inductien and growth of callus in anthers of sev-

eral sweet potato cultivars. In the present study, the frequency of

callus formation from stem and tuberous rool explants was also

very high. These resu]ts suggest that 2,4-D could induce calli at

high frequencies from any kinds of explants. Cel] suspension eu]-

tures ceu]d be established from al1 ef callus cultures (Fjg. 4a).

Table 8. Effect ef water treatment on protoptast yield.

CultivarsYield (eellstg fresh weight}

Control Watertreatment

Beniazuma

Benihayato

Chugoku2S

Tosabeni

8.6xIoS71

× te"7.8

× le`58

× loS

3.5× 10`1.5

× 10`1g

× 1o"1.2

× lo'

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26

Bulletin of Ishikawa Agncultural College No. 26 (1996)

tMv:ptt)tz.'.g..#Wxs'.

aV-e

a.

k

eeeeza

dec

fae

.ss. gs ntk #.

g, Callus and roet formation from protop[ast of mesophyll and

ulturedce]ls

Ce]1 suspension cultures of sweet potato Bur = 60 pJn

Frcsh[y isolateci mesephyl] protoplasts. BaT' = 40 ym.

Freshly tsolated protop]asts ot' cultu[ed cells Bai = 70 pm

Firstdivisionofprotoplastaftcr3daysofeulture.Bar=30pm.

Protoptast-demved visible colonies in the liquid medium after40

days ef cuilure Bar = 50 pm

Protoplast-derived ca]1i plated on gelian gurn-solidified medium.

Bar=1Oinm

Advcntitious root formation from mesophyll piotoplast-derived

callus Bar = 1Omm

phyll and calAus cells protoplasts were transferred ento the regen-

eration media, a fow foots werc fermed from the cat]] placed on the

media containing both BA at O.5, 1.0 or 2.0 mgtl and IAA at O.?

mg/] (Fig. 4g), but no shoets were produced. No ofganegenesis

was observed in suspension cu]ture-deri ved protoptasts, which rnay

suggest that the suspension cutture had ]ost their rcgeneration abil-

ity during the long period of cu]ture.

Root development from the caUi fas a]so been reported m those

derived from petio]e protoplasts (Bidney and Shepard 1980). A]-

though, shoot regeneration from protoplast-derived ealli was

achieved in sweet potato (Murata et at, 1986, Sbihachakr andDucreux1987,PereraandOzias-Akins1991,Belarminoetal.1994),

the frequency of shoot in these studies is stilHow. In exper]ment 2

of Chapter 2, 1 describe the efficient sheot preduction from adven-

titious roots ol' L trichocany)a. It seems that shoot can also be re-

generated from adventitious root at a high frequency in lpomoeaspccies, through which regeneration of shoots from protoplast-de-

nved roots wil[ be achieved

The isolation of protoplasts from cultured cel]s such as ca]]us

and ce]1 sugpension cultures has both advantages and disadvantages

when compared to isolation of protoplasts from intact plants. Cul-

tured eeli could offcr a number of culturable protoplasts, In the

present study, the division freguency of protoplasts derived from

cultured celas was Iw]ce that of mesophyll protoplasts. However, it

may be ditTicult to regeneratc any oreans from cultured cell-de-

rived protoplasts due to Iose of totipotency during the subcu]ture,

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27

M Otanr 1Apphcationofplantbiotechnologyforbreedingoflpomoeaspecies

The culture method which enab}e to maintain the totipotency of

cultured cells for ]ong time has to be develeped for further investi-

gation on somatic hybridization of sweet potato

Chapter 4Genetic transformation of lpomoea genus

Experiment 1 : Transformation of sweet potato (ipomoea batatas (L.) Lam.) plants by Agrobacterium rhizogenes

Introduction

InfecuonofwoundeddicotyiedonousplantswithAgiobactertum

rhtzogenes can cause hairy root formation at the site of infection

Newly developmg tissues are charactenzed by the acquisition ofa

bacten al DNA fragment from the Ri pl asrmd ofA thtzogenes (White

et al 1982) The [[LDNA fragments ofRi plasmid are stablyinte-

gratedintoplantchromosomes(Chiltonetal 1982) Regenerated

p}ants have been obtained after Iransformation with R]-T-DNA in

agronomically irnportant species such as alfa1fa (Spano et al 1987),

caulifiower {David and Tbmpe 1988), eilsced rape <Ooms et al1985), potato (Oome et al 1985, Hanish Tbn Cate et al 1988) and

tomato (Shahin et al 1986) The plants regenerated from the hairy

roots were characteflzed in several species by distin¢ ti ve morpho-

]ogica] abnormalmes such as wnnk1ed Ieaves, $hortened internodes,

reduced apica] dominance and abundant, frequently branching roots

that showed [educed geotropism (Spano et al 1987, David and

'[bmpe 1988, Ooms et al 1985, Oome et al 198S, Hamsh lbn Cate

et al 1988, Shahin et a] 1986, tcpfer]984) Such phenotypic al-

terations could be of interest for breeding programs

Sweet potato (ipomoea batatas L (Lam )) is a megor crop, but

the formation of transformed plants in this species has not beenreportcd Here 1 report hairy root formation and plant regeneration

in sweet potato transformed by A rhizogenes I also descfibe the

transgenic plant harbonng both nptiI and gus genes introduced by abinary vector system based on the A rhizogenes-Ri p]asmid

Materials and Methods

Plantmaterials

Feurteen cultivars of sweet potato (lpomoea batatas (L ) Lam ),Bise, Chugoku 25, Chugoku 35, Hi-starch, Kanto 18, Kanto 94,

Koukei l4, Kyukei 17-3043, Naeshirazu, Nonn 2, Okinawa 100,

Shinya, Yamakawamurasaki and W5 1 were used [[b ebtarn in vitro

ptants of sweet potato, menstem culture was performed accordmg

to the procedure of Otani et al (1987) In vitro plants were grown

on LS medium containing O 25% (WIV) gellan gum (Kelce Divi-sion of Merck & Co tnc ) and no growth regulators at 260C under

contmuous illuminatien at 38pmel m'! s" from daylight fiuorescent

tubes

Bacterial strains and yectors

Agrobactenum rhtzogenes, one agropine type strain C15834)

and seven mikimopine (lsogai et al 1988) type strains A5 (MAFF02.10265), Al3 CMAFF02-10266), H4 (MAFF02.10267), C8

(MAFF02-10268), D6 (MAFF02-10269), NIAES 1724 (MAFF03-O1724) and NIAES1725 (MAFF03-O1725) (Daimen et al 1990)

were grown for 16hr at 27'C in a 1iquid YEB medium (Vervliet et

al 1974)

ITie binary vector plasmid pBI121 (Fig 5, Jefferson et al 1987)

was rnobihzed from Eschertchta cott C600 to A rhizogenes C8,

harbonng a wild mikimopme-type R: ptasmid, by tnparental mat-

ing using pRK2013 as a helper plasmid (Dittaet al 1980), and cul-

tured under the same conditions

Transformation and establishment ofhairy root cultures

The apical third to fifth of fully expanded leaves of in vitro

plants were inoculated with A, thtzogenes, accordmg to the meth-

ods of Noda et a} (1987) The inoculated leaf disks were placed on

sterilized moist paper in a glass petm dish and incubated at 26eC in

the dark After 3 to 5 days of incubation, leaf disks were Irans-

ferred to a 1% (W/V) agar or O 32% (WIV) gellan gum-so]]difiedLS medium supplemented w]th 500yglml Clafbranop(Hoechst) and

mcubated under the same condMons Bactema-free root 1ines were

obtained after excision of single roots and propagauon on LS me-

dium supplemented with 400pgtmt CtaforanO and O 32% (WIV)gellan gurn durtng three subcultures

Plant regeneration from hairy roots

The hairy roots <30 to 40 rnm in length) of sweet potato were

transferred onto O 32% gellan gum-solidified LS medium ]acking

both antibiotics and growth regu]ators, and cultured at 26eC under

contmuous i]]umtnation at 38umol m'i s'i by dayhght fiuorescent

tubes The percen{age ofhairy roots with shoot formation was cal-

cuaated by the number ofhairy roots with shoet formation per thenumbcr of hairy roots transferred to the regenefation mcdium

Detectien of opines (agropine, mannopine and mikimopine)

The op:nes in both hairy roots and ]eaves of regenerated plantsfrom hairy roots were detected by si]ver-staimng for agropine and

mannopine (Petit et al 1983), and Pauiy reagent-staining for

mik]mopme after paper electrophoresis aecording to the method of

Petit et al CPetit et al 1986)

DNA isolation and Southern blot hybridizatio"

lbtal DNAwas isolated from lcaves ofin vitro plantby sodium

dodecyl sulfate {SDS) extraction method accerding to Honda and

Hirai (1990) DNA digested with EcoRI was sutljected to electro-

phoresisandtransferredtoAmersham'sHybond-Nnylonmembrane

Southern analysis was earried out usmg the TL-DNA probe, pLJ 1

"984), with digoxigemn (DIG) labelltng and AMPPDR (Tropix,lnc ) detection system (Boeh-nger Mannheirn) accordmg to the

suppher's instructions The DNA ofhairy root-derived plants trans-

formed by the mikimopine-type strain ofA rhtzogenes were also

analyzed by the method of Handa (1992)

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28

Bulletin ef lshikawa Agricultura] College No26(1996)

Resistance of hairy root deriyed plants to kanamycin

Shoots of hairy root-demved plants were excised as 20 to 30

mm long segments, and transplanted onto O 32% (WIV) gellan gum-solidified LS medium supplemented with 1oo mgll kanamycin The

plates were mcubated at 260C under continuous il}umination at

38umol m'2 s'i from daylight fluorescent tubes

(;US assay

The GUS acuvities ef leaves and roots ef regenerated plants

were determined by the method of Jefferson et a[ (1987)

Polymerase chain reaction (PCR) analysis of introducedforetgn

genes:

For analysis of the plants regenerated from the hairy roots by

polymerase chain reaction (PCR), the sequence of two genes, NosP-

uptll-NosT and 35SP-gus-NosT, was ampldied with the following

sets ofpnmcrs Pnmer PNosP (5' AAA TGC TCC ACT GAC Grl'l'

CC 3') is located in the NosP region, 81bp 5' of the translation ini-

tiation site (AIrG), and pnmer PNosT C5i CGC AAG ACC GGC

AAC AGG AI' 3') is complementary to the sequence ef the 3Lflank-

ing region, 80bp 3' of the Nos translational stop signa] (TAA) Pmmcr

P35SP (S' GAI] GTG MA TCT CCA CTG AC 3') is located in {he

CaMV35S promoter region Acombination of the primers, PNosP

and PNosT was expectcd to produce a 1 5SObp product in the NosP-

trptJJ- NosT A combination ofthe pnmers, P35SPand PNosT was

expected to amplify the 2kbp region in the 35SP-g"s-NosT Fifty

nanogram$oftemplateDNAand200nMofeachprimerwerem]xed

with 5ul of 1O× Tbq DNA polymerase buffer (SOOmM KCI, 1oomM

Tns-HCI {pH 8 3), 15mM MgCl, and O 1% CWtV) gelatin), plus100yM of dNTP mixturc (fmal concentration) and 1 unit of Taq

DNA polymerase (Perkin Elmer Cetus-fakara) in a total volume ef

50E 1 Thnty cycles of PCR were perfbrmed in a programmed tem-

perature control system (PC-7oo, Astec, Ibkyo), in which a single

cycle consisted of the fol!owing steps denaturation at 940C for ]

minute, annealing at S5"C for 2 minutes, and DNA synthesis at 72"C

for3 minutes Amp]ified DNAs were analyzed by cth]dium bro-

mide (EtBr) stammg after 1% (WIV) agarose gel electrephoresis

by the method of Handa (1992)

Septualcrosses

Two transgemc Chugoku 25 plants Cone was transt'ormed byA

thtzogenes strain A13, and the other was by the strain 15834) and a

non-transformed Chugoku 25 plant were crosses with non- trans-

formed Yamakawamurasaki plants The seeds wcre immersed in

97% <vlv) sulfunc acid for 30 minutes, nnsed three times with ster-

ile disti]]ed water, and wefe placed on LS medium supplemented

with O 2S% {WtV) gellan gum Cultures were kepl at 26'C under

contmuous illumination at 38 pmol m'Z s'i frorn day}ight fluores-

cent Iamps Phenotypes in morpho]ogy and opine synthesis of the

progemes were determined after 3 weeks of culture

Resuks

Pathogenicity ofA. rhizogenes strains

Hairy roots were first observed within seven days after inocu-

lation (Fig 6a) They were produced from the wounded sites, for

example, from the cut surface ef excised petiole Mikimopine type

strains ofA rhtzogenes gave similar pathogentcity to agropine type

strain, 15834 Thepereentage of]eafdisksproducing hairy rootsin

cv Chugoku 25 was 22 8% in l5834, 20% inA5, 25% mA13, 25%

m H4, 26 7% m C8, 40 5% in D6, 31 l% m NIAES]724 and 20%

in NIAES1725 On the other hand, genotypic difl'erences in hairy

root formation frcquency werc observed arneng thc cu]tivar) tcsted

when leafdisks were mocuiated with A thtzogenes stram A l 3 (Ibble9) Chugoku 25, Kanto ]8, Koukei 14 and Shmya produced hairy

roots at a lew frequency (23 5-27 8%), while the other cultivars

produced hairy roots at a higher (51 4-88 6%) frequency

Plant regeneration from hairy root

Adventitious sheot formation was observed when hairy roots

were cultured on O 32% geHan gum-solidified LS medium lacking

both ant]biotics and growth fegu[ators under continuous illumina-

tion (Fig 6b) Regeneration of complete plants was obtained in 5

cultivars (Chugoku 25. Chugeku 35, Hi-slarch, Kyukei ]7-3043

and Yamakawamurasakt) among 1Ocuttivars tested The percent-

ageofhairy roots with shoot format]on was, 50% for Chugoku 25,

]OO% for Chugoku 35, 80% t'or Hi-starch, 40 9% for Kyukei 17-

3043 and 41 7% fer Yamakawamurasaki These findings suggest

that there are vanetal ditferences in shoot regeneration from the

hairy roots Shoot formation was not irnproved by the addit]on of

BA (O 5 and 2 mgll) to regeneration medium (data not shown>

Opine synthesis by hairy roots and hairy root-derived plants

Both agropine and mannopine were detected in the extracts of

thc hairy roots and ieavcs of the p]ants fegenerated from the hairy

roots obtained by infection with the agropine-type strain of A

rhtzogenes Mikimopine was atso detected in Ihe hairy roots and

leaves of the plants regenerated from Ihe hairy roots obtained by

the infectson with mikimopine-type bactenal strains{Fig 7) Sincc

Table 9 Varielal drft'erences in hairy root format]on inciuced by

A rhtlogenes MAFF02-]0266

Cu]tlvars No leafdisks mocu]ated

No teafdisksforming hal ry roots

BiseChugoku25

Chugoku 35

Hi-starehKanto ;3Kanto

94Koukei 14Kyukei

17-3043

Naeshirazu

Nomn2Ok]nawa1OO

ShmyaYamakawa;nurasaki

wst

317330373.4333678353g31343540

(9,)25 <8C} 6)tS

<24 7)13 C43 3)25

(67 6)

9{26S)19

c5. 7 6)IO

C27 8}52 (66 7)tS (51 4>2S

(6S 8)I8 (5g l)

8 (23 5)3] (88 6}30 (7S O)

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29

M OtaniApphcation ef plant bioteehnology for breeding of lpomoea species

NOS-fto NFr II NOS-ter CalviV35-Promoter t9-Glucuren]dase(GUS) NOS-terLB

RB

Fig SStructure of the bmary veetor pBI 121

the opme synthcsis ofA rhtzogenes-inoculated plants is encoded

by T-DNAof the Ri p]asmid (Chilton et al 1982), the present find-

ings mdicate stable mamtenance and expressien of TLDNA in these

hairy roots and the plants regenerated from them

Morphology of plants regenerated from hairy roots

The adventitious shoots formed on the hairy roots were excised

and placed on O 8% (WIV) agar so"ddied LS mcdium The trans-

planted shoots grew and Tooted well, and these plantlets were trans-

ferred te pots contaimng a 2 1 (V/V), vermiculite and per]ite mix-

ture These potted plants were maintained at 26bC under a 16-hour

per day photoperiod in a growth chamber for 30 days, and the plants

transformed by the rmkimopine-type strains ofA rhtzogenes were

transplanted to the expenmental field of tshikawaAgncu]tural Col-

lege Ail ofthese plants survived

Al] plants regenerated from the hairy roots showed Ri T-DNA-

induced morphological changes Aemal parts showed decreased

apical dominancc and shortened internodes (Fig 8a) The average

stem length was shortened about one-fifth of that of the normal

plants A difference in stem length was observed among the

transgenic ptanls regenerated from the independent hairy root 1ines

The average stem length (.mean ± standard deviation) of the trans-

formed plants of cy Ylamakawamurasak"nduced byA rktzogenes

A13 vamed from 23 ± 1O 6 cm to 104 ± 7 7 crn, while that of the

untransformed plants was 225 2 ± 1O cm The leaves were wnnkled

and smaller than normal (Fig 8b) The shape of the fiower was

changed dramatical]y , hairy root-deayed p]ants had small and star-

shaped flowers (Fig 8c) Pollen fertdity ofhanry root-demvedplants

was not a]tered tn comparison to normal sweet potato plants, since

more than 90% of po]len grains of both of the hairy root-derived

plants and untransformed plants were stamed by O 5% acetocarTnine

solut]on Subterranean parts showed abundant roots with exten-

siye branching and sma]ler storage roots (Fig 8d) Interestingly,

the numbers of storage roots in the plants regenerated from the hairy

root was equal to that of norrnal plants The average fresh weight

of thc storage roots from the plant regenerated from the hairy roots

was 3 6 g, while that of a normal plant was 39 4 g

The storage roots of both hairy root-denyed plants and normal

plants were incubated at 28"C to allow adventMous shoots to sprout

from them In the storage roots ofhairy root-denved plant formed

fow adventmous shoots and the shoots grew more slowly than those

produced from the storage roots ofnormal plants, while the storage

roots ofthe former showed excessive rooting with intensive branch-

ing (Fig 8e)

T-DNAanalysis

One ofthe regenerated plants, SE3-5, which was confirmed to

contain both

agropine

and

mannopi.n.

g, .w..i .s further

analyzed

to

con-

firrn the transformation ofRi-[fl.-DNA (Fig 9) Ne hybndlzationsignal was observed between the TL-DNA probg and DNA from an

untransformed plant On the contraryi--thg.PNA of a regenerated

plant hybridized to the same probe and contarngd four mternal frag-

ments comigrating with EcoRI fragments of th'e probe (EcoRI-40,15, 36, and 37a+b) Th]s regenerated plant also had eight border

fragmentscorrespondingtoJunctionsbetweenTL-DNA(EcoRI-3a

and 3b) and plant DNA llhis suggested that at least 4 copies ofTL-

DNA were presen"n the regenerated plant, SE3-5.

DNA from hairy roet-derived plants transformed by the

mikimopine-type strain ofA thtzogenes hybndrzed to the T･DNA

probe, 7 5kbp EcoRI fragment including the core TLDNA region

Handa 1992), while no hybndization signal was observed between

the same probe and DNA from the untransforrned plant

Fig 6a-b Plant regeneratton from hairy root ofsweet potato transformed by Agrobactertum rhizogenev a

byA rhtzogenes Bar = 20 mm b Sboot formation from hairy roet transformed by A rhtzogenes

':t

Harry root formation from leafdisk moculated

Bar=10mrn

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IshikawaAgricultural

30

College

Bul]etinot'Ishikawa Agnculturai College No26{1996)

Fig7 Opine assay of extracts of five regenerated plants denved from

independent hairy root clones ofcv Ytimakawamurasaki

Tlen mtcrohters of extract cerresponding to approximately 50 mg of

leaves(freghweight)weres]nttedonAdvantecNo2filterpapcr(lbyo

Roshi. Inc ) and subJected to etectrophoresis at 20 Vtcm Lane M.

standard miknnopine, lane 1, 2. 3. 4 and 5, five indcpendei]t

regenerated plants. Jane H, standard histtdtne Upperarrow indicates

hist]dine and lower arrow [nd]cates rmkimopine Mikimopine was

not detected in untransfoiined plants (lane C). but was observed in

halry root- denved plants (lane 1-5}

Transgenic sweet potato plants possessing nptll and gus genes

Six ptants were regenerated from difterent hairy root ciones ot'

cv Yamakawamurasaki induced byA rhtzogenes C8 possessingabmary vector, pBl]2l Al] regenerated ptants produced miktmepine

Shoots of thesc rcgencratcd plants were transferred to LS medimn

con{ainmg 100 mgtl kanamycin Three of the regenerated p]ants

produced many roots 3 days aftcr transfer, whi]e the other plantsdid not forrn any rools Jn our prelirninary study using ha] ry roets

transformed by wild type A rhi`:ogcnes, no root format{on or root

growth was observed on the medium contain]ng K}O rngll kanamy-

cin Thus. these three p]ants which formed many rootg on kanamy-

cin-contaimng medium werc defincd as being resistanE to kanamy-

cln

Root tips and lcafdisks of rcgenerated plants were staincd with

5-brome-4-ch]oro-3-indolyl- glucuromde (X-gluc) for 16 hours to

detect the cxpressien ot' the gus gene

Gcnomie DNAs from each regeneratcd p]ant and untrans fenned

p]ant were subyected to PCR amplificanon as a rapid check for trans-

tormaoon by T-DNA ofpBlI21 Hairy root-demvcd p}ants gave

the predicted PCR bands for nptll and gus genes, while

untransformed plants were negative for both products {Fig 1O)

ln the present study 50% (316) of haJry roots obtained werc

doubly transformed by Ri plasmid TLDNAand pBI121 TLDNAwith-

out any se[ectlon pressure

Inheritance of the Ri transformed phenotype

The gefmination rate was 84 6% - ]O09, There were no clear

drfferences in the germination The progeny which denved frorn

the crosses between the transgemc plants and untransformed

Yamakawamurasaki plants segregated Ri-transformed phenotype

About 70% of these progeny demonstratcd typical hairy root traits

FigSa-e Morpho]ogical abnormatit]es of

regenerated planl transformed by A

rht`'.ogener

a Stem oi' p]ant regenerated t'rorn hairy root

transformed by A rhizogenev A13 (tower)

and untransformed ptant (upper) of sweet

potato cv Chugoku 25 Morphologlcal

abnorrnalit]es such as smal]er leaves,

shortened internodes and reduced apical

deminancewereobservedmthetransfonned

plant Bar = 30 mtn

b Wnnk]ed ]eaves observed in transformed

ptant Bar = 30 mm

c Ftowers of transferrned ptant {rlght) and

untransformed p]ant (teft) Bar = ]5 mm

d Aemal paris(upper) and subterranean parts

{]ower) of transi'erined plant {]efO and

untransfermed piant {-ght} C,

untransfermed plant, T, transformed plant

Bar=20cm

e Adventitieus shoot formation from the

storage root of untransformed (]eft) and

transformed plant (nght) Bar := 20 cm

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31

M Otani' Application ofplant biotechnology for breedmg od' lpomoea species

Fig9

t .t t t.tttt t tt. t#tt ttttt t.

.,.･･,:･. ttt t t:

t .t t.

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ipt,-

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.t ･tt tt t.t ' .. . ..ww..t ttt ttt t･t . Tl4- t, t.- t

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Southern b]ot analysis of EcoRI digested DNA from regenerated p]ant

transformed by A rhizogenes IS834

Digested DNAs were subjeeted to electrophoresis in a O g% agarose

geA, bloted onto a nylon filter, and then hybndized against the pLJ 1

probe which was labeled with non-radioactive digoxigenin- dUTP

usmgaDNALabehngandDitectionKit<BoehnngerMannhelm)N,

untransformed plant, T, transformed plant Arrows indicate possib]e

border fragments A physical map of the TL-DNA digested with

EcoRI is illustrated below Intaet TLDNAs are expected to contain

Eeo15 (4 3kb), 36 {1 8kb). 37 (1 6kb) and 4e (1 4kb) fragments

present in pLj1 Eight bands observed in the transformant are bordier

fragments which are luncnons between TtDNA (Eco 3a or 3b) and

the adJacent plant DNA

tttt

.t

1

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'

'

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el

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tttt't t t/ttttt/ t

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'''

ttttttttttt ttttttttt.tttttt.

" .tt tttttttttt tttttt ttt ttt. ttttt t 'tt

t ' tttt.

'ttt

/t './ttt . 'mosT

LB

asmamewwma--nt･ .t tt - -t .tt f5kbp

' -t tttttt

''

.. -t t tt

zekop ...,

t t

Fig 1O PCR expenment on DNA samples ffom 3 kanamycin resistant plants

showing GUS activity PCR amplifieation of the nptll gene and gus

gene

EtBr staining pattern after agarose gel electrophoresis of PCR

arnplified samp]es Lane t, XHindlll DNA size standard (23 1, 9 4,

6 5, 4 3. 2 3, 2 O and O 5kbp fragments from top to bottom), ]ane 2,

ptasiTud pBI 121 showmg the 1 5kbp expected band forthe aptll gene

or the 2kbp expected band- for the gus gene ,lane 3, untransfermed

plant,lanes4,5arid6,threeindependenttransformedplants

such as shortened mtemodes, reduced hypocotyNength and wnnkIed

leaf Ihe segregation data from beth crosses were consistent with

two independent, dominant Ri TiDNA loci (fable 1O)

Discussion

In the present study we report successful transformation ofJ

hatatas mediated byA rhizogenes Diffcrences in pathogen]city

among vanous wild type A rhtzogenes strains were observed The

pathogenicity of mikimopine-type strains was similar to that of the

Table 1O Segregation ofthe Ri tfansformed phene{ype ]n progeny

ofcrosses between two transl'ormed Chugoku 25 plants

andacu]uvarYamakawamurasaki

cross No of germlnatlonseeds

phenotype

Ri type'i normatx2va]ue*2

untEaasfcrrrmdplantXTamakamemaasaku

Al/11'iXYamhwammwhd

ISS341"XYannskawamurasaki

(%)

13 ll( g4 6)

9 9(100 )

26 2S< 96 2)

o717 tl28O

04nsO65ns

*i Ri type Ri plasmid-transformed type

*2 Segregation ratio lested was 3 1

ns Not-slgnlficant*3

Transgen]e plant A13-1 was transformed by A rhizogeneT A13*4

Transgemc plant [5834-l was transformed by A rhizt)genev strain 15834

agropine-type strain Interestingly, a mannopme-type strain,

ArM1 23 isolated in Japan gave the highest rate ofhairy roet forma-

tion {57 6% of]nfected ]eaf explants produced hairy roots) in our

prcltmmary study David and Tlempe (1988) reported that agropine-

type strains (A4, IS834) were more yirulent than mannopine-type

strains (8196, TR]Ol, TR7) My present fmdings suggest that

mannopine-type strain, ArM]23 ts the mest virulent, although the

viruleney of the mikimopine-type strains is similar to that of the

agroplne-type straln ln sweet potato

Genotypic differences in the susceptibility among the cultlvars

of sweet petato to tnfection with A rhizogenes A13 were also ob-

served S]milar findings have been reported in kale (Chnstey et al1992). faba bean (Ramsay et al 1990). soybean (Owens and Cress

l985) and potato (De Vnes- Uutewaa] et a] 1988) In addition, the

vanetal differences were fbund ]n shoot regeneration from the hairyroots Shoot format]on was not improved by the addition of BA

<O 5 and 2 mgt1) or zeatin (2 mgh) to the regeneration medrum (datanot shewn)

Al] thc charactenstics observed in the transformed sweet po-tato plants were similar to the deyiations observed in the corre-

spondmgorgansoftransformedConvotvulusarvenstsCIbpfer1984),

a species of Convotvutaceae family

The choiee ofA rhtzogenes was made to allow the possibihty

of using the morphological alterations produced by the Ri-TLDNA

for sweet potato breeding programs In fact, the aenal parts of trans-

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Bulletm of Ishikawa Agrrcuttural Co]Iege No26(1996)

formed plants showed shortened internodes Since dwarfness is

one of the important charactenstics in crop breeding, shortening of

internodes in transformed p]ants is adesirable change On the other

hand, the subterrancan parts of transformed plants showed abun-

dant roots with extensive branching and smaller storage roots as

descnbed in this paper Recent]y introduction of the rotB gene or

the rol C gene of R]-TZ-DNA to a p]ant celi resulted in transgenic

ptants with morpho]ogica] abnormalmes such as reduced apical

dominance and shortened internodes (Oono et al 1987, van Altvorst

et al 1992) The transgenic tomato plant possessmg only the rol B

gene was charactenzed by a reduction in both internode length and

apica] dommance, while the root system of this transgenie plant

was simi]ar to that ofuntransformecl plants (van A]tvorst et al ]992)

The subterranean parts of the transgemc sweet potato plant with the

rol B gene or rot C gene shoutd be compared with those ot' transgenic

plants with the intact Ri plasm]d T-DNA introduced

The present study a[so $hewed that A rhti.ogenes can be used

successfully to transfer foreign genes from a b]nary vector into sweet

potato plants Although t'ew reports have been publi$hed on p]antregenerauon from protop]asts in the sweet potato (Murata et al l987,

Sihaehakr and Ducreux 1987, Perera and Ozias-Akins l991), the

proloplast culture technique has not yet been fu]ly established to

apply to the direct gene transferin sweet potato Since whole p]ants

can be regenerated at a high frequency from hairy roots transformed

by A ihizogenes, the binary vector syslem based on the Ri plasmid

could be cfficient]y app]ied for the geneuc transforma"on el' sweet

potato

Experiment 2 : Fertile transgenic plants of ipomoea trichocarpa Ell. induced by different strains of

Agrobacteriumrhizogenes

Introduction

Many dieotyledonous plants infected with Agrobactenum

rhizogenes are known to show the deve}opment of hairy roots at the

inoculation sites, wherc T-DNA, a bactenal DNA fragment trom

the Ri plasmid ofA thizogenes is incorporated into host p]antce[[(White et al 1982) and stably integrated jnto plant chromosomes

(Chi]ton et a] ]982) The p]ants regenerated from thehairy roots

were characten7ed in scvera] species by dist]ncnve morphotogical

abnorma]ttEes such as wnnkled leaves, shortened internedcs, redueed

apical dominance and abundant roots with extensive branching and

reduccd geotropism (lbpfer l984, Ooms et al 1985a,b. Shahin et

ai l986, Spano et a] 1987, David and Tempe 1988, Hanish Tlen

Cate et al 1988) Such phenotypic alterations could be of [n:erest

for horticultura] crop breeding programs

The genus lpomoea contains an impor!ant tuber crop, sweel

potato (l batatas (L)Lam)and severa] ornamental crops such as

Caire mormng glory U cairtca L ), Mrs Horsfall's morning glory

{J hor,stintkae Hook ) and btue morning glory (I tncolorCav ) I

trtchocarpa Ell , a wild relative of sweet potato, is a]so cxpcctcd {o

bc ut]1ized as a new ornamental crop Tb broadcn the genetic vari-

abi]ity of these species, it may be intcrestmg to incorporatc the genesinvoivedmILDNAofA thizogenes lnthisgenus,however,whole

pl ant regenerat]on from hai ry roots transformed by Ri- plasmid had

only bccn reported inswee{ petato (Otani et al 1993) and no trans-

formed plants by A rhizogenes have been produced in wild rela-

tives of sweet potato ln the present study, we report the productionoftransgenic plants of J trtchocarpa by A rhtzogenes through hairy

root formation and ptant regcneration and the inheritance of the

transformed traits to thc progenies I also describe the prediuction

of transgenic p]ant harboring both nptU and gus gene by using a

binary vector system based on theA riulogenes-Ri p]asmid

Materials and Methods

Plantmaterials

Seeds of lpomoea in('hocarpa Ell were immersed in 97% (VlV) sulfuric ae;d for 30 mmutes, nnsed three times with slenle dis-

ti1]cd water, and placed on LS medium (Linsmaier and Skoog 1965)

supplemented with O 25% (W/V) ge]lan gum ( Ke[co Division ofMerck & Co Inc ) Cultures wefe kept at 26'C under continuous

i[lumination at 38ymol m ! s

i by daylight fiuorescent tubes

Bacterial strains

lbnw]]dstrainsofAgrvbactenumrhizogenes,oneagropinetype

strain, ]5834 cPetit et al ]9S3), enemannopine type strain, ArMl23

(Otani et a] 1993), one cucumop]ne lype stra]n, NCPPB 2659 (Pctit

et al ]986) and seyen mikirnopine Usogai et al 1988) type strains,A5 <MAFF 02. ]0265), A]3 {MAFF02.10266), H4 (MAFF02-10267), C8 (MAFF02.I0268), D6 (MAFF02. 10269), NIAES1724

(MAFF03-O1724) and NIAES1725 (MAFF03-O1725) (Daimon et

al 1990) were used forthe study An agropine-type slram, 15834

harboring the binary vceter pla.smid pBI12l which possesses the

chimenc neomye]n phosphotran$ferase (nptll) and B-giucuronidase

(gus) genes (Otani ct al ]993) was a]se used for the transforma-

tion Thcy wcre grown tor [6hr at 27"C in a 1iquid YEB medtum

(Very[iet et a] 1974) before us]ng for the infect]on

Induction,decontaminationandestablishmentofhairyrootcul-

tures

1] n days after sewing, cotyledons were excised from in vitre

secdhngs and ]nocu]ated withA rhiz.ogenes. according to the meth-

edsofOtani et a] (Otani et a[ ]993) The inoculated cotyledons

wcre placcd on steri1]zed mo]st paper ]n a gtass Petri dish and incu-

bated at 26℃ jn the dark After 3 days of incubation, cotyledon

exp[ants w'ere transferred to aO 32% ge:an gum-so]idified LS me-

dium supplcmented with 500pgim] Claforan'" CHoechst Japan Ltd )and incubatcd under the same eondMens Bactema-free root 1ines

were obtained after exc]s]on ol' sing]e roots and propagation w!th

three subcultures on LS medium supplementcd with 400pg/ml

C[aforan'" and O 32% (W/V) gellan gum

Plant regeneration from hairy reots

The hairy roots C30 to 40 mm in length) were transferfed onto

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M Otan:App]ication ef plant biotechnology for breeding of lpornoea species

O 32% (WIV) gellan gurn- solidified LS medium lackmg both anti-

biotics and growth regulators, and cultu red at 26"C under eontinu-

ous illummation at 38 umol m'! s'i by day]ight fluorescent tubes

After 30 days of culture, the percentage of hairy roots with shoot

formation was calculated by the number ofhairy roots with shoot

formation per the number of hairy roots translerred to the regenera-

tion medium

Detection of opines

The opines were detected in the ]eaves of regenerated plants

fremhairyrootsbysilver-stamingforagropineandmannopine(Petst

et al 1983), and Pau}y reagent-staining for eucumopine and

rnikimep]ne after paper electrophoresis according to the method of

Petit et al (1986) [lbn microliters ofextract correspondmg to ap-

proximately 50 mg of ]eaves (fresh weight) were spotted on

Advantec No 2 filtcr paper (R]yo Roshi, Inc ) and subjected to elec-

trophoresis at 20 Vlcm

Fig 11 Harry root formation from excised cotyledons ef ipomoea

trtchocarpa inoculated withAgrobactertum rhtzogenes Al3 Bar =

]5 inin

was morc than 80% in all bactenal strains

Phenotypic characterizatien of hairy root-deriyed plants

The hairy root-derived plant}ets were transferred to pots con-

taining a 3 1 (VtV), vermiculite and perhte mixture These potted

plants were mamtained at 260C under a 16-hour photopenod in a

growth chamber for 30 days, and the plants transformed by the

mikimopine-type strains ofA rhtzogenes were transplanted to the

expenmental field and analyzed to determ]ne vamous phenotypic

charactcristics such as stem length, mternode length, petiole length,

size of teaves, shape and size of fiowers, fiowenng time and po]len

fert]1ityResistance

of hairy root-deriyed plants to kanamycin

Shoots of ha]ry root-denyed plants were dissected tnto 20 to 30

mm ]ong segments, and transplanted onto O 32% <WIV) gel1an gum-sohddied LS medium supplemented with 1OO mgA kanamycin in

90× 15 mm plastic Petri dish The plates were incubated at 26"C

under contmuous illumination at 38 vmol m'! s'i from day]ight fluo-

rescent tubes

Histochemical GUS assay

The GUS activmes of ]eaves and roots of regenerated plants

were detected by stain]ng with S- bromo-4-chloro-3-indolyl-glucu-

ron]de (X-gluc) at 37'C for l6 hours according to the method ot'

jefferson et al (1987>

Results

Shoet regeneration from hairy root

Adventitious shoot formation was observed when hairy roots

were cultured on O 32% (WIV) ge]lan gum-solidified LS medium

lacking both antibiottcs and growth regulators under continuous il-

lum]nation The pereentage of hairy roots with shoot formation

was different among the bactenai strains, 96 2% (51 / 53) in l5834,

50% (5/lO) m ArMi23, 50% (511O) m NIAES1724 and 63 8% (37158)inA13,respectively

Opine synthesis by hairy roots and hairy root-deriyed p)ants

Both agropine and mannopine were detected in the extracts of

the hairy roots and leaves of the plants regenerated from the hairy

roots obtamed by mfection with the agropine-type strain ofA,

rhtzogenes Cucumopine and mikimopine were also detected in

the hairy reots and leaves of the plants regenerated from the hairy

roots obtained by the infection with cucumopine- and mikimopine-

type bacteria] strains, respectively Smce the opine synthesis ofA

rhtzogenes-moculated plants is encoded by T-DNA of the Ri plas-

Table l1 Rh]zogenic response of coty}edon in lpomoea tnchocarpa

after infectson with 1O strarns ofAgrohactenetrn thizogenes

Bactenatstrains No cotyledon

exp]antsinoculatedNo

cotyledon explants

forming hanry roots

Hairy root induction

Hairy roots were first observed w]thin seven days after inocu-

]ation They wefe produced frem the wounded sites such as the cut

surface of excised petio]e (Fig 11) There was no difference in

hairy root formation between leat and coty]edon exp]ants {data not

shown) Tbb]c 11 shows the percentage ofcotyledon explants pro-

duc!ng hairy roots in each bacterial strain No clear difference in

pathogenicity was observed arnong yanous w]]d type A rhtzogenes

strains The percentage ofcotyledon explants producing hairy roots

IS834ArMl23NCPPB2659

A5A]3C8D6H4NIAESO1724

NIAESO1725

59353737372527373S2e

(%)55(

93 2}

35(100 )

37(100 )30( 81 1>

34( 91 9)

25(100 )

24( 88 9)

34( 91 9)

32( 91 4)

20(100 )

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Bulletin of Ishikawa Agncultural Co]lege No26{1996)

Fig 12 Rowers of plants regenerated frem hairy roots ofl trtchocarpa

transformed by five strains ofA rhizogenes {A-E) and untransformed

plant (N) A. 15834 {agropine strarn), B, ArMI23 (mannopine strmn>,

C, A13, rnikimopine strain, D, NJAES1724 (rmkimopine strain), E.

NCPPB2659 (cueumopine stra!n) Bar =40 mm

c" T

Fig 1 3 Var]avon m flower morpho]ogy arnong the transfomied l trtchocarpa

piants by A riu7vgenev tS834 T, fleweFs of 1] independent

transformed clones, C. a contro] flower ofuntransformed p]ant Bar

=40mm

mid <Chi]ton et a] ]982), the prcsent findings indicate stable inte-

gration and exprcssion of ILDNA in these hairy roets and the plantsregenerated from them

Morphology of plants regenerated from hairy roots

A]1 of thc plants transformed by the mikimopinc-type strains of

A rhizogenes could survive after transplanting to the expenmenta]

field A]1 the plants regenerated from the hairy roots showed R] IL

DNA-induced morpho]ogical changes In the two hairy root-de-

nved p]ants, A13-l which was transformed by A13, and 1724-1

which was transformed by NIAES1724, the average stern ]ength

was less than one-Ihird of that of the norma] p]ants, the leat' size and

petiole [ength reduced (Tlable 12), and the leaves of transformed

plants were crmkled

The rnorphology of flowers also changed dramatica]ly in hairy

root-dcrived plants The flowers ot'Al3-1 and ;724-] wcre sma]],

reeurved and star-shaped (Table 12), while those of two indcpcn-

dent transformed plants induced by 15834, an agropme type strain

were slightly smaller than those ofuntransformed plants (F]g ]2)

Somc fiower buds (8 2% of fiowers in A13-1 and 23 9% ]n ]724- ])

of mikimopine-type Ri plasmid-mediated transformed p]ants did

not flower, and th]s phenomenon was not observcd in the plantstransformed by other bactena] stratns The hairy root- denved plantstransformedbyacucumopine-typestra]n,NCPPB2659hadtheflow-

ers with similar size and shape to those ofthe plants transformed by

themikimopine-type strains The flowers ofthe hairy root-denved

p]ants transformed by mannopine-type strain, ArM 123 were differ-

ent frorn those transformed by other bacterial strains A]1 the plantstransformcd by mikimopine type strains exhibited invariab]e fea-

tures of the transformed phenotype, while the yamat]on in fiower

shapes was observed among the transformed plants by agropine-

type strain, ]5834 (F]g 13) In both A13-1 and 1724-1, the first

fiowering date delayed more than 7 days and the number of flowersoftransformed plants decreased as eompared to the untransformed

plants (Table 12) However, pollen fetu]]ty of hairy root-defived

p]ants was not altered in cempanson to the untransfonned plants,

sincc more than 90% of potlen grains of both ef the hairy root-

der]ved plants and the untransformed p]ants were stained by O5%

acetocarmine solution In my preliminary study, both oftwoinde-

pendent R]-transformed p]ants by l5834 did not show delay in fiow-

ering, but the number of flowers of those transformed plants de-

crcased as compared to the untransformed p}ants (data not shown)

Tab]e 12 Companson of stcm, leaf and fiower phenotypcs on

transgenicanduntransfbrmedplantsoflpomoeatrrchtxatpa

's'Iem

Lmes iength

ctmi.

Table 13 Inheritance of the Ri transformed phcnotype in selfed

progeny of transgenic lpomoea trtchocarpa

Sizcofthe5thexpanded

]eavcs{c:n)t/'len'gth

' "･,dth pet/o:e:EnLIE''

S]zeof]eavesCtTn}" Fmst No of Poilen

nowenng flowers" feailTcy

daLc

1]neNo o[' plants

lengthw]dthTotatR] lypc'Nomal=!value (3 ])

po

AB-] 117±4!12 ±os2i ±o4 i6+05:6+03 1!±04 Aug!q !.6)'5 9Sl

17!4-1 147B33#'04 21±OS li+03. 26tOG 3;t.04 Aug IU VI 994

Control 4JS=r 3S 60±eS SI±07 34+OS 29t{}2 40102 Au! 2] 6,S40 9Si

- The stem ]ength was measured from the top of the stem to the base ef

the 5th fu[Ly expanded leaf*2 incan '.L' S D*;The number of f]owers were counted for 29days frorn thc first

flowemngdate

A13.1]724.]ArM123-]IS834.]i5834-215834-34t735554103S6 345729437S41 7t6t6142515137O37267OOIO03O10

i Ri type Ri plasmici-transt'ormed phenotype

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M Otani ,Applicataon of plant biotechnoiogy for bTeedmg of lpomoea species

ttt

- ･ -.･: .. .･

?Xaggetewt,eel,es xiSx

Fig 14 Opine assay ef Ieafextfacts ofsix selfed progenies ofa transformed

l trtchocatpa plant, 1724-1 Lane M. standard mikimopine, lane C,

untransformeci plant, lane T, a transformed plant 1724-1 , Iane 1-6.

six selfed progenies of the transformed plant 1724-1 The arrow

mdicates mikimopine Mikimopine was only detected in the hairy

root-demved plant (lane T) and some of its progenies (lane 1, 2, 4

andS)

The frequency of seed set in these transgcmc plants was 94 - 94 6%,

while that of the controls was 94% No c]ear differences in the

number of seeds per pod was a] so observed between the transgenic

plants (3 7 ±O 5 - 3 8 ±O 5 (mean± standard deviation)) and the con-

trols (3 9±O,3) Subterranean parts of hairy root-denved plants

showed abundant roots with extensive branching and plagiotropic

growth

Inheritance of the transformed phenotype

Six Ri-transforrned plant lmes (1S834-1, 15834-2, 15834-3,

ArM123-1,A13-l and 1724-1) were sclf-fetuhzed and the passageof Ri ILDNA through meiosis was observed Seeds were obtained

from al1 of the six transformed plants There were no differences in

the seed gerrnination between the untransformed plant and the prog-

enies of the Ri-transformed plants The progenies which showed

Ri-transformed phenotype also exhibited opine synthesis (Ftg 14)Beth Rr- transfbrmed phenotype and opine production were segre-

gated together into these progenies In all cases, the x2 values cal-

culated from the segregation ratsos were not significantly different

at O OS level from the expected ratios of 3 . KThble 13), indicatingthat the Ri plasmid T-DNA was integrated at a single srte on the

chremoseme and the transformed phenotypes and opine produc-

#k'"Si;lggpa.

e,#i･igl･

ewi,

ttt ttt tt .ttetiii{sl'il#

':t{

l' ite s, i, .: i, t"

ee sg:::nv .ltS. t t.t g...,･,.. /, t. t ttttt･ tt tttt"t . ttttttt tt t ttttttt t ttttttt tt. ttttt.sbttttt

e?:: ,;.=t:tt;:f:;

t t.-tt .tt ttttt tt t

tt

ttt

gggkgss,lifi.,#,.x#;.;syi,,i:.,i,g-i･;g.gi･i･Ii{･l･#･

Fig 15 Detection ofGUS act:vity in transformed l trtchocarpa plant by

histochemical assay transformed leaf {T> turned blue by staining

with X-gluc, while untransformed leaf (C) drd not show any stalnmg

tionwereinhentedasMendekandommanttrmtsmthese6transgemc

plants

Production of transgenic plants possessing uptU and gus genes

TXuenty plants were regenerated from different hmry root clones

induced by A rhtzogenes 1 5834 possessing a binary vector, pBI121

All regenerated plants produced agropine and mannopine, and ex-

hibited hairy root syndrome Shoots of these regenerated plantswere transferred to LS medium containing 1OO mgtl kanarnycin

Six of the regenerated plants produced many roots 3 days after trans-

fer, whi]e the other plants did not form any roots In our prelimi-nary study using hairy roots transformed by wild type A rhtzogenes

strain, 15834, no root formation or root growth was observed on

the mcdium containing 1OO mgA kanamycin Thus, these six plants

which formed many roots on kanamycin-containing medium were

defined as being resistant to kanamycin Root trps and leafdisks of

the regenerated plants werc stained with X-gluc, suggesting that

gus gene was integrated and expressed in these transgenic p]ants

(Fig 15) Consequently, 30% (6120) ofhairy root-demved plantsobtained in the present study were doubly transformed by Ri p]as-mid TLDNA and pBI121 T- DNA without any selection pressure

Seeds obtained frem four selfifetu1ized transgenic plants were

Table 14Inhemtance of the transformed tralts in progeny obtained by self pollination of

four transgenic lpomoea tnchocarpa ptants

transformedphenotypestiCbu-squarctestS]

stramsNo seedlmgsRiphcnotypsKmS]resistanceandGUSacteviry

Rl+ 1!1+ Rl+ 121- Rl-12]+ Rl-t2T-1 integratson 2integfation kntcgratron 2integratio] C3b {t5 1) (3b {15 1)

NGTNG2NG3Nca S7I03S64341784]19 Deo! oooe 1425l52 oeloe]Ol9)'

±sOt9

OOIO03OlS65to6g

*i Phenotype R!+, Ri phenotype Ri-, no Ri phenotype 121+, kanamycin reststanl and i?-

glucuronidase positive t21-, kanamyein sensittve and iV-g]ucuronidase negative*2

Chisquare test statistically sigm fieant at O 05 ]evel (*) and Oel level (**).] Km kanamycin

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Bulletm of Ishikawa Agncuttural Cotiege No26(1996>

gerrninated and the seedlings wcre analyzed for the introduced traits

The genetic analysis showed that segregation for both the Ri phe-

notypes and the pBI121-derived phenotypes includmg kanamycin

resistance and GUS-activity occurred in all the strains (Tabte 14)In strains NGI

- NG3, both Ri- and pBI121-denyed phenotypes

segregated as single dominant genes However, the data from strain

NG4 suggested that two groups ef phenotypes were segregated in-

dependently as the genes located at two dom] nant loci

Discussion

In the present study we succeeded in the transformation of t

tnchocarpa mediated by A thtzogenes No c]ear differences in

pathogemcity were observed among vanous w]ld type A thizogenes

strains The mannopine-type strain,ArM123 and the mikimopine-

type strams, A5, A13, H4, C8, D6, NIAES 1724 and NIAES 1725,

which were isolated in Japan, have a similar virulency to that ot

agropine-type strain when inoculated to I trtchocarpa Although a

similar result had been observed in our prcvious study on sweet

petato (Otani et al l993), the frequency of hairy root fermation in

J trtchocarpawas much higher than that ef sweet potato On the

other hand, difference in pathogenicity ha$ been observed in many

other bactenum-p]antcombinations David and Tempe (1988) re-

ported that agropine-type $trains (A4, 15834) were more virutent

than mannopine-type strains (8196, TR ]O1, TR7) Dobigny et a[

(1995) observed that rhizogenic respense vaned with the baeter]a]

strams, when potato stem explants were inoculated with

cucumopine-type strain 2659, more than 7e% produced hairy roots,

while few rhizogenic responses (2-17%) or no response at all were

obtained with the agropine-type strain, 15834 and mannopine-type

strain, 8196 These results suggested that lpomoea species may

have a w]de susceptibility to varieusA nhtzogenes strains

Shoot regenera"on from hairy roots is an another important

prerequis]te for the successfu1 production of transgenic p]ants by

using A rhtzogenes One of the main reasons for the successful

production of transgenic J tnchocatpa plants with the chimenc

nptll and gus genes in the prescnt study might be the selection ofA

rhizogenes strain, 15834, which showed the highest frequency of

shoot regeneration among the bactemal strains tested

All the chafacteristics observed in the transformed J tnchocaeJa

plants were similar to those observed in the transformed plants ef

Convotvutus arvensts (lbpfer ]984) and sweet potato (Otan] et al

1993), both of which belong to Convotvulaceae fam]ly In the

present study diffcrence in fiower shapes was obseryed among the

plants transformed with different bactena] strains (Fig 12) as wetl

as among the plants transformed with one agropine-type strain,

15834 (Fig 13) Simi]ar fmdings havebeen reported in Convolvu-

tus arvensts and tobacco ([fepfer 1984), potato (Hanisch Tbn Cate

et al 1988), Stytosanthes hetmtlts (Manners and Way 1989) and sweet

potato (Otani et al l993) Dwarfness of the aena] parts was a]so

observed in all ofthe transformed plants Since altered flower shape

and dwarfness are Ihe useful charactefisncs for J trtchocatpa, it is

possible that the transformed plants cou}d be used as the gene sources

for the breeding of this species with novel traits

For furthcr breeding of these Ri-transformed plants, it is impor-

tant that they have ferulity in sexua] organs Berthommeu and

Jouanin (1992) reported that all the transformed rapid cycling cab-

bag¢ plants by A rhizogenes A4 were female feftile, but a]most

male stemle Fortunately, the Ri plasmid-mediated transformed

ptants obtai ned in thc present study exhibited good seed product:onby selfing due to male and female fetu1ity Consequent]y, the trans-

tormed phenotypes were sexuat[y transmitted to the progentes in I

tnchocany)a The transformation system established in the present

study wil[ give us the possibi]ity to introduce some ]mportant for-

eign genes ]nto this spec]es such as disease [esistance and novel

flower color genes ProducLion of pure hnes with dwarfed plants

and novel flower type are now tn progress

Chapter 5General Discussien

Recent development of ptant bio{echnologies sueh as sematie

hybr:dization and genetic transfermauon ha$ been providmg new

approaches for plant breeding These new techniques atlow to in-

troduce novel Iraits into erop speaes, which are not possible to trans-

fer by conventional eross- breeding and mutatien-breeding meth-

ods Success of these technolog]c$ was init]a]ly 1imited to the

Sotanaceous species inve] ved ]n the genera such as Mcottana, Pe-

t"ma and Solanum Cetl and genetic engineering techniques h3ve

suecessfu]ly been apphed for important crop species such as nce,

maize and Brassica species in the 1980's Ihrough l990's, and sev-

eral somattc hybrid plants and transgenic plants have already been

produced m these species Howevef, these techno}ogies have not

been developed we][ in sweet potato and other lpomoea spec]esbecause of the lack of an efficient and rcproducib]e p[ant regenera-

uon systcm ]n these lpotnoea spccies Tberefore,I f]rst examined

to estab]ish et'[lcient p[ant regeneratton systems ]n sweet potate and

lpornoea trtchocau)a in cxpcnmcnt ] and 2 tn chaptcr 2

Plant regenerat]en trom eu]tured tissues [s influenced by mainly

two factors One isachemica] factor and the other isaphysica] one

relating to culture condit]ens (BhoJwani ttnd Razdan 19g3) Thc

importance ol' chemical factor was tirst suggested by Skoog and

Mil[cr (]957) who proposed the eoncept that the relative concen-

tration of auxin and cytok]nin dctermines organogenetic d]tTeren-

tiatien in plant tissue culture Since then, numerous studLes have

been suggest)ng the imporTance of kind, combinatien and concen-

tration of p]ant growth regu]ators In this study, theretore,Iexam-

ined rnainly about chernicat factors

For the plant regeneration from cu]tured tissues, it has been

suggested that selection ot denor tissues is important (O'Hara and

Street 1978, Sh]mada 1978, Flick et al l983, Pienk 1987) In the

present study on cultivar Chugoku 25, leaf t]gsue-der]ved cal]i gavea higher frequency of shoot regeneratmn than thc cdlli demved from

stem and petiole Iissues ]n thc previous studies on other cultivars,

however, either on]y spontancous or low frequcncy plant rcgcnera-

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M Otani' Apphcatson ofplant biotech"otogy for breeding of lpotnnea species

tion was observed from leaf hssue-denved callus cultures irrespec-

tive of the vanous attempts of exogenous hormonal control (Carswelland Locy 1984, Kobayashi 1984, Suga and Inkura 1988) In the

present study, the eptimum concentraUon of BA in the medium for

the adventitious shoot formation from leaftissue- denved callt var-

ied among the different genotypes These results suggest that the

level ot' exegenous cytokmin required for shoot forrnation depended

ontheamountofendogenouscytokininintheculturedtissueswhich

must vary among the different genotypes

In $ome of the recalcitfant ttssues, adventitious shoot forma-

tion couid be mduced or promoted by apptying abscisic acid

(Yamaguch] and Nakajirna 1974, Shepard 1980). and etylene in-

hibitors such as aminoethoxyvinylglycine (Robmson and Adams

1987, ChJ et al 1990) and silver nitrate (Purnhauser et al 1987,

Songstad et al 1988, Chi et al 1990, Chraibt et al l991) ln the

present study, addition ef abscisic acid and silver nitrate to the cal-

lus induction medium clearly promoted shoot productien In my

preliminary study using l tnchocarpa and I trijida, shoot regen-

eration was ebtained ongy from the calli induced on the medium

containing abscisic acid These findings suggest that abscis]c aeid

and ethylene are invo]ved in the production of the calli which pos-

sess the abihty of high shoot regenerauon These fmd]ngs also sug-

gest a possibility for overcoming the low abi]ity of plant regenera-

tion of Ihe other recalcitrant species m lpomoea genus

Another alternative way to regenerate ptants from cultured tis-

sues in recalcitrant plant species might be to induce shools from

adventittous rools regenerated frorn cultured tissues Shoot difier-

enttation from root exp]ants has been reported in some ptant spe-

cies such as Petunta h.vbrtda <CohJin et a] 1979), L.vcoperstcon

peruvianum {Norton and Boil l954), Stylosanthes gu.vamensts

(Mei]er andBroughton 1981), Pelargontum spp (Skirvin and janick

1976), Eustoma grandoflorum {Fu[ukawaet a] l990) and so on ]n

the present study, the frequency of shooE regeneration from adven-

titious roots of I tnchocarpa was very high, and similar results

were obtained in other lpomoea species (Carswell and Locy 1984,

Liuet al 1990, Belarmino et al i992) Asadventitiousroot forma-

tion is easier Ihan shoot regeneration in general, shoot ditTerentia-

tion from adventitious roots a]so be app]ied to other reca]citrant

plantspeaes

In many plant species, somactonal variation was observed

arnong the regenerated plants CBajeq 1990) Altheugh some of the

genetic changes are expected to ofi'erusefu] traits forplant breed-

ing programs, megonty of the changes are uncxpected use]ess ones

and occur very often Therefore, somaclonal vanation shou]d be

suppressed in biotechnological studies on the plants of particular

genotype In the present study, onty one regenerdnt ofsweet potatowas morpho]ogica]ly abnorma] among regenerated plants ot sweet

potato and I trtchocarpa Smcethe frequency of the somaclonal

vanalion seems to be re]auvely tow, these ptant regeneration sys-

tems may be useful for genetic transformation study

Production of sematic hybrids is usefu] for sweel potato breed-

ing as the means for introducing the novel traits from sexuatly in-

compatib]e genotypes or species As the first step for somatie hy-

bndizauon, I examined protoplast isolation and culture in sweet

potato in expenmcnt l and 2 in chapter 3 lsolation of protoplasts

from mesophylt tissues and cel] cu]tures, and callus forrnation and

root regenefation from the protoplasts were achieved in the present

study However, roet regenefation from protoplast-denved cal]i

was vcry low Recently, efficient plant regeneration systems from

protoplasts were developed usmg fast-growing embryogenic cell

suspension cu]tures as the source material in cereals such as maize

(Pnoh and StndahH989, Rhodes et al 1988, Shillito et al 1989),

rice (Fuj]mura et al l985, Tbnyama et a] 1986, Kyozuka et a]

1987) and wheat (Harr]s et al 1988, Wang et al i990, Vasi] et al

1990), whieh had been reca[citrant plant species for a long ttme It

is, therefore, expected that eslab]]shment and the use ot embryo-

genic cell suspension cultures may also help to overcome the iow

frcquency of organogcnesis trom protop]ast-derived calli in swcet

potato

In Japan, ene of the important disease of sweet potato is sweet

potato feathery mottle virus (SPFMV) This virus causes the dis-

colorauen in skin of sterage roots, [n all of culuvars, espeaally m

Kokei 14, one ef the mosT important cultivars in Japan As a resu]t,

the market value ot the roots is senously reduced (Usugi et a] 1991)

At present, there is no way of introducing virus resistance character

Io this cultivar by traditiona] breeding method Therefore, virus

free plants produced by the memstern tip cu]ture have been sup-

phed to farmers, who must buy new virus frec p]ants every two to

three years because the plants should be reinfected with the v]rus

by aphids as soon as they are grown in the fie]d Thus, it is neces-

sary to develop new technology wh!ch can introduce the yirus re-

sistance trait to this cu]tivar In ethcr plant species, it has alrcady

bccn repoited lhat transgenic plants which cxpress a viral coat pro-

te]n gene were often resistant te infection with the virus from wh]ch

the coat protein sequcnee was denved (Sturtevant and Beachy 1992)

Recently, Mon et al (1995) isolated thc coat pro tein gene of SPFMV-

S Therefore, estab]ishment ot an efficient system ofgenetic trans-

formation has been expected tbr ]ntroducing this virus resistant gene

into sweet potato As descnbed in expenment 1 in chapter 2, thc

protoplast cu]ture technique m sweet polato hab not yet been fu11y

established fer app]y to the direct gene del!very at present How-

ever, in some species of lpomoea genus, it seems that adyentitious

shoots may read]]y regenerate from adventitious roets Thus, I ex-

amined geneuc trans[brmation of sweet pota[o and I trtchocarpa

usi ng Agrohac/terrum thtzogenes ]n experiment l and 2 in chapter 4

and succeeded in obtaining several transgenic plants Furtherrmore,

these transgemc p]ants were tertile and Ri plasm)d T-DNAs and

introdueed [ore]gn genes (nptll and gus) were certainly transmitted

to their progenies ]n the transformation by direct gene transfer to

protoplasts, transgenic plants obtained frequently showed stenhty

CRhodesetal 1988,Tbfiyamaetat 1988) Therefore,A rhizogenes-

mediated transtormation should be superior to the direct DNA de-

1ivery mto protoptasts for obtaming ferti3e transgemc plants Fur-

thermore, this transformation method does not require any special

]nstruments such as etectroporator device and part]cle gun t'or gene

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38

BullettnofIshtka-'aAgncu[turalCe]legeNo26(t996)

delivery te target celts Reeently, a number ef transgenic ricc and

ma]ze p[ants were ohtamed by Agrobactertum mediated-transfer-

mation method (Chan et al 1993, Hiei et aT ]994, Saito et aH 995)

These resu}ts suggest that the host range of this bactemum is much

wider than previously expected and that the possibility of transfor-

mation of monocotyledonous important crops mcluding hortieut-

tural plants such as 1]ty and orchid with un efficiency similar to that

oftransformat]on]ndicoty]edons

Anether advantage ofA rhtzogenes-rneciiated transformanon

is that w]ld type Ri plasmid 'P

DNA posses$ed by this bacte"umcan cause vamous physio]ogieal and morphologtcal alterations on

rccipient plants Some of these altcred traits might be appked to

improve the agronomica] eharacter ot hort]culturat plants

Pellegrmeschi et al (1994) succccded in improv]ng the ornamental

,t,,ua}ity and tfagrance production ef lernon geramum through ge-net;c tfanstormat]en by wild tlr･peA rhtzosenes Transgen]c plantsof both snapdragon (Anttrrhtnum mcijus) and prairte gentian

(Eustoma grandiflorum) showcd the increase in number et flowers

which may be useful for improvmg hortrcultural character of these

plant species (Handa 1992a, Handa ]992b) Godo and MiK]991)

reported thattransgen]c plants of Nieremhergta sctoparia exhibtted

dwarfness without any horticu]turally negative characlers In the

present study, transgenic p]ants of both sweeL potato and i

trtchocarpa showed var]ous alterations in physielogical and mor-

phological phenotypes Espec]a]ty, dwarthess ]n both plant species

and a] tered flower morpho]ogics in l trtchocarpa may be ot impor-

tance as useful ag"cultura] chdractcrs These resu]ts suggest that

the Ri p]asmid ILDNA ofA ritii.ogenes might otteT new traits tor

horticutturat crop breeding

Acknowledgement

For aecomp[]shing et this thesis, tu$ my great plcasurc to firstly

acknowledge the helpofProf Masahire Mii,Chiba University. and

Prof Taktko Shimada, Ishikawtt Agrtcu]tural College, -ho have

abetted and aided teaLhing whenever they could

Thanks are also due to help to Prot' Kyuya Harada, Prol Shiro

Kunta,Asso Prol' 'fakato

KobaandAsso Prof YoshttoAsano for

kindly mspeclion ofthis thesis I am gratfu1 toAsso Prot Hiroyuki

Daimon,UmversityotOsakaPfefeeture,forthcgiftofrmk]mopine

strains ofAgrohactertum rltizogenes and to Dr Mitsuteru Ohta,

Agmcultural Expemmentai Stat]on ofShtLuoka Prefecture, for thc

g]ft ol'Agrohactertum rhizoeenesArMl23 I w]sh to thank Pref

H]roshi Kamada, 1lsukubaUniversity, Prof Tbtsuro Murata, Kyushu

Tbka] Univers]ty, Dr Keiko Ishikawa, Chiba University, Dr 1lakash]

Handa, Tsukuba Umyersity, Dr Masako Komaki, Agmcullural Ex-

periment Station of lsh]kawa Prefecture, Mr Hiroyeshi Teruya,

Agncultura] Expemmental Station of Okinawa Prefecture. Mr

Hiromasa Sawada, Horucultural Expenmentai Station of Kochi

Pret'ecture, Prol' Mitsuo Imura and Asse Pref Satosh] Oke,

Ishikawa Agncuttura] Collcge and Asso Prof Hisato Kunitake,

Kyushu Tbkai University foruseiul advices and expenmental help

about plant moleeujar biology and tissue cul{ure ( am atso greatlu]

to Dr Muneo hzuka, Dr Hiroo Niizeki and the late l)ref Kunikazu

Uek], the former presidcnt of Ishikawa Agncultural College, for

giving me courage to study this thcsis

l indebted to Mr Tbshinari Godo, Mr Keichi Nemoto and all

members of Research lnsti tute ofAgricultural Resources, Ishikawa

Agricu]tural College for all facrhties and suggestitons provided

Abbreyiations

ABA Abscistc acidBA 6-benzy]adcnine

2.4-D 2,4-dichlorophenoxyaceuc acid

EDIA ethylenediamme letracetie acid

gus B-glucuromdase

IAA indole acetic acid

LS medium Linsmmer and Skoog [nediumMES 2-N-morphohnoethanesulfonicaad

Nes nopahne synthase

nptii neoirtycm phosphotransfcrase

PGR Ptantgrowth regulater

X-glue 5-bromo-4-chEore-3-tndo]yl glucuromde

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エshikawa 　 Agrioultural 　 College

43

MOtam 　Applicatton　ofplanl 　biotechnology　for　breeding　of　lpomoea 　species

サツマイモ属植物の育種におけるバイオテクノロジー

の応用

大谷基泰

‘附属農業資源研究所　植物細胞育種研究室丿

摘　要

　近年，細胞融合や遺伝子導入などバイオテクノロジーが，

トマトやイネなどの作物の育種に応用され始めて，いくつか

の成果が出てきている．オレンジとカラタチの体細胞雑種の

「オレンジカラタチ中間母本農 1号」やアメリカで遺伝予組

換え植物として始めて売り出された「Flavr　SavrTM」はその主

要な成果である．しかしながら，園芸植物種を多く含む lpo−

moea 属植物では，バイオテクノロジーに関する研究がイネ，トウモロコシ，ジャガイモなどの主要作物に比べて大きく立

ち遅れている状態である．本論文では，加oπ］oea 属植物の中

で，サツマイモと Itnchocarpa についてバイオテクノロジー

を利用した育種の可能性について論じた．

　本論文は，第 1章の序論から第 5 章の総合考察まで，全 5

章から構成される

　第 1 章の総合序論では，本論文の背景と目的，さらにハイ

オテクノロジーの植物育種への応用の可能性について例をあ

げて述べ，Jpomoea属植物種の育種におけるハイオテクノロ

ジーの重要性を論じた

　第 2章では，ハイオテクノロシー技術を確立する際の最も

基本的な技術である培養組織からの植物体再生について検討

した　その結果，サツマイモとその近縁野生種 ∬ tnchocarpa

の葉片由来カルスからの効率的な不定芽の再分化条件が明ら

かになった．

　サツマイモ品種中国 25号の葉片由来カルスからの不定芽

形成は，培養組織からの再分化の際に広く用いられている

BA の添加によっては促進されず，再分化培地としては植物

生長調節物質を添加しない LS培地が適当であった．その際，エチレン阻害剤である AgNO3 をカルス誘導培地に 2　mg ！］の

農度で添加することによって極めて高い不定芽形成率を得る

ことができた　このことから，サツマイモではカルス誘導時

のエチレンの発生を抑制することによって再分化能を持った

カルスを誘導することができることが示唆された不定芽形

成は ABA によっても影響され，2　mgfl 　ABA をカルス誘導培

地に添加して得られたカルスから高い頻度で不定芽が再分化

した．

　　1　tnchocarpa の葉片’由来カルスからの不定芽形成は，再

分化培地に BA を添加することによって促進することがで

き，サッマイモの場合と異なった傾向を示したこのことか

ら，∬ trJchocarpaは内生サイトカイニンの量がサツマイモと

比べて低いと考えられた．また，∬ trichocarpaの場合，カル

スから不定芽を得るのには，カルスから直接不定芽を誘導す

る方法と，カルスから再生 L た不定根を，LS ホルモンフリー

培地に移植して不定根から不定芽を誘導する二通りの方法に

よって可能であった．Ipomoea属植物では，カルスからの不

定根分化は，不定芽の分化に比べて比較的高頻度で生じるの

で，この不定根を経由した不定芽の再生方法によって，他の

1ρomoe 嘱植物のカルスからの再生系を確立することの可能

性が示唆された．

　第 3章では，細胞融合やプロトプラストへの遺伝子導入と

いったハイオテクノロジー技術の基礎となるプロトプラスト

の単離と培養についてサツマイモの葉肉組織と培養細胞を材

料にしておこなった．その結果，葉肉組織からのプロトプラ

ストの単離には，ln・Vltro 植物の展開葉の切片を，滅菌水に約

16時間浸す前処理を行うことが有効であり，前処理を行わな

かったものに比べて 20倍以上の収量が得られた．葉肉プロ

トプラストと培養細胞由来プロトプラストは同様の比較的簡

単な培養方法によって，効率良くカルス化することが可能で

あり，プロトプラスト由来カルスからの不定芽の形成は見ら

れなかったが，不定根の再生が観察された．

　 rg　4 章では，野生型Agrobactenuni 　rhizogenes によるサツマ

イモと ∬ trJchocarpa の形質転換を行った．その結果，ミキモ

ビン型のハクテリアをサツマイモ数品種に接種した実験で

は，毛状根形成について品種間差異が認められ，さらに，サ

ソマイモ品種中国 25号に異なった系統のバクテリアを接種

したところ，ハクテリア間でも毛状根形成に差異が生じるの

を確認できた　このことは，供試する植物材料に適したバク

テリア系統を選択する必要性があることを示唆しているサ

ッマイモでは，ミキモピン型のハクテリアによって比較的高

頻度に毛状根を誘導することができた　これに対して，1

tnchocarpa では，バクテリア系統間での毛状根形成に著しい

差異は認められず，全てのバクテリアにおいて 809。以上の切

片から毛状根が形成された毛状根を植物ホルモンを含まな

い LS 培地に移植することによって，サッマイモと 1

trichocarpaの両種の毛状根から不定芽を再生させることが可

能であった．再生した形質転換体は，葉が波打つ，地上部が

矮化するといった Rl プラスミドで形質転換した植物体に特

微的に見られる特性を示したサッマイモでは，地上部の矮

化は，単位面積当たりに栽植できる株数の増加につながり，このことは単位面積当たりの収量の向上につながるために有

用な形質と考えられた　’tnch （rcarpa の形質転換体は，地ヒ

部の矮化とともに花や葉の形態的な変化が本植物種の園芸的

な価値を向上する Lで興味深いものであった．これらの形賑

的な特性は，生殖によって安定して後代に遺伝することが確

認できた　さらに，R1 プラスミドを用いたバイナリーベク

ター

法による両植物種への外来遺伝子（gus，　npt 　Jl遺伝子）の導入を試みたところ，形成された毛状根のうちサツマイモでは

50％が，そして 1trichocarpaでは 30％が外来遺伝子を保持し

た毛状根であり，それらの毛状根から外来遺伝子を保持した

植物体の再生も容易になされた．以上の結果から，野生型の

R1 プラスミドによるサツマイモと 1　 trtchocarpa の形質転換

は，これら二種の植物種に新たな形質を付与するという点で

有用なものであることが示唆された．さらに，Rlプラスミド

を用いたハイナリーベクター

法はサツマイモと∬ mchocalpa

への外来遺伝子の導入法として有用であることが確認され

た

［キー

ワー

ド］ lpomoea 属，形質転換，サツマイモ，組織培

　　　　　　養，ハイオテクノロジー

N 工工一Eleotronio 　Library 　

Application ptant biotechRology - J-STAGE

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