J Japan sOc_Hort.Sci.64(4):919-925. 1996.

Production of Interspecific Hybrids between Liliwm regale and

L. rubelluln vra Ovule Culture

Yoshiji N五mi,ⅣIasaru Nakano and Ken‐ichiro Maki*

F“%′りげ4g蔵%ιιππ,」Vじじgα′α υπづυι7Stり,2-8θ5θルα7aSんじ,Nじじgα′α95θ-21

Summary

Lil ium regale and L. rubellurn were crossed reciprocally to introduce economically andhorticulturally desirable traits of L. regole, such as vigorous growth and disease resist-ance, into L. rubellum.

1. In l. regale xL. rubellutn, seeds containing an embryo were obtained with a low fre-quency (3.3 %) by stigmatic poll ination at anthesis, but they did not germinate in soil orin vitro. Seedlings were successfully obtained with frequencies of 5.3 to 6.7 7o by ap-plying ovule culture 30 to 60 days after poll ination.

Z.ln L. rubellumxL. regale, the growth of pollen tubes was inhibited in the style afterstigmatic poll ination at anthesis; hence, no ovule with an embryo was obtained. Growth ofpollen tubes was promoted by stigmatic poll ination when the poll ination was done 2 to 5days after anthesis. Embryos definitely formed if stigmatic poll ination was carried out 5days after anthesis, but they could not be rescued even when ovule culture was uti l ized.Cut-style poll ination had no effect on production of ovules with an embryo.

3. Hybridity of seedlings resulted from l. regalexL. rubellum was confirmed by nu-c lear rDNA analys is . Al l o f the hybr id p lants analyzed were d ip lo id wi th 2n: 24chromosomes'and showed pollen ferti l i t ies of below 3 7o. Flower color of the hybrids waslight pink. A single hybrid clone produced a double flower.

Introduction

L . rube l l um, gene ra l l y ca l l ed 'H imesa -yu r i ' o r'Otome-yur i ' , is ind igenous to Ni igata, Fukushima,

and Yamagata Pref . , Japan (Shimizu, 1987). This

species has potent ia l as an ornamental crop be-cause of i ts beaut i fu l , t rumpet-shaped p ink f lower,p leasant f ragrance, and dwarf ism. Therefore, somegrowers have at tempted to produce bulbs commer-

c ia l ly . However, bulb product ion has not been suc-

cessful because L. rubellum is very sensitive to

fungal d iseases and into lerant of h igh tempera-

tures during the summer. On the other hand, L. re-gale , a species nat ive to China, has desi rable

traits such as vigorous growth and resistance to

v i ra l and fungal d iseases. Thus, at tempts to in t ro-

duce these t ra i ts in to the other species are inp rog ress (Van Cre i j e t a l . , 1992 ) .

Received for publication 31 August 1995.* Present address: Japan DelMonte Corp., Ltd., 3748

Shimizu-cho. Numata 378.

In the gents Lil iurz , interspecific hybridization

has been used to incorporate d isease res is tance

and adaptabi l i ty , as wel l as f lower colors and

forms from one species to another. These pro-

grams have proved to be successful in developing

nove l cu l t i va rs (Van Tuy l e t a l . , 1988 , 1991 ) . Be -

cause hybridization between L. regale and L. rubel-

lwm has not yet been reported, we attempted and

succeeded in producing in terspeci f ic hybr ids v ia

ovule cul ture.

Materials and Methods

1. Plant materiols and pollination erperiments

Potted plants of. L. regale and l. rubellurn were

grown under natura l , forc ing or retard ing-cul ture

condi t ions to adjust f lower ing t imes.

Flowers of female parents were emasculated one

day before anthesis. In most poll ination experi-

ments, f resh pol len was used, but in some cases,

pol len stored according to Ni imi and Shiokawa

919

920 Y NHmi,M NakanO and K Maki

( f 992) was used. In L. regale xL. rubel lum, s t igmat-ic pol l inat ion, in which pol len was appl ied ontothe st igma, was carr ied out only at anthesis . In areciprocal cross, l,. rubellurn xL. regale, stigmaticpol l inat ion was carr ied out 1 and 2 days beforean thes i s , a t an thes i s , and 1 , 2 ,3 ,4 , and 5 days a f -ter anthesis . In the la t ter cross, the cut-s ty le pol -

l inat ion technique as descr ibed in a prev ious pa-per (L i and Ni imi , 1995) was a lso per formed 2days before anthesis , at anthesis , and 5 days af teranthesis .

2. Ouule culture

Ovar ies co l l ec ted I0 , 20 , 30 , 40 , 50 , and 60days af ter pol l inat ion were sur face-dis infectedwi th 70 % ethanol for 1 min, wi th a commercia lb leach solut ion conta in ing 1.8 7o chlor ine for 10min, and r insed three t imes wi th s ter i l ized, d is-t i l led water . Thi r ty , 40, and 50 days af ter pol l ina-

t ion, ovules were excised f rom ovar ies and cul -tured on a medium composed of B-5 macronutr ient(Gamborg et a l . , 1968), a hal f s t rength MS micro-nutrient (Murashige and Skoog, 1962), amino acidsby P rakash and G i l es (1936 ) , 0 .1 mg . l i t e r - r NAA,5 % sucrose and 0.7 o/o D,gar at pH 5.7. For ovanesharvested 10 and 20 days af ter pol l inat ion, ovary-s l ice cul ture pr ior to ovule cul ture was used be-cause ovules were too smal l to be excised. Ovary-s l ice d isks were prepared by cut t ing ovar iest ransversely in to sect ions 5 mm th ick and p laced

wi th the basal cut end down on the medium. Onemonth af ter inoculat ion, the enlarg ing ovules wereexcised f rom cul tured ovary-s l ice d isks and sub-cul tured onto a f resh medium. Al l cu l tures beforeembryo germinat ion were kept at 25"C and in thedark; the germinated ovules were p laced undercont inuous i l luminat ion (1000 to 1 2 00 lx) sup.p l ied by whi te f luorescent lamps.

3. Obsenations of pollen tube growth and embryo de-uelopment

To trace pollen tube growth in the style afterst igmat ic pol l inat ion, s ty les were f ixed in FAA(formal in : acet ic ac id : 70 Vo ethanol : 5 :5:90) ,s ta ined wi th a 0.1 % cot ton-b lue solut ion for about40 m in (N i im i , 1991 ) , and then d i ssec ted . Po l l entubes were observed under a l ight microscope, andrelat ive length of pol len tubes as a percentage ofsty le Iength was recorded as previously descr ibed(Li and Ni imi , 1995). Development of embryos af -

ter pol l inat ion was examined h is to logical ly on se-r ia l , longi tudinal , paraf f in-embedded sect ions(Wakizuka and Nakaj ima, I97 5) .

4. Ribosonal DNA (rDNA) analysis

Tota l DNA was iso lated f rom leaves of pot tedplants us ing a sodium dodecyl su l fate (SDS) ex-t ract ion method according to Honda and Hira i(1990). Iso lated DNAs were d igested wi th rest r ic-tion enzymes and separated on agarose gels fol-lowed by blotting onto nylonmembrane fi l ters(Hybond-N, Amersham, UK). Analyses of rDNA us-ing a non-radioact ive DNA label ing and detect ionki t (Boehr inger Mannheim, IN) were per formed bythe method previously descr ibed (Nakano and Mi i ,1993). The DNA f ragment conta in ing the ent i rerDNA sequences of r ice (Takaiwa et a l . , 1985)was prepared f rom plasmid pRR217 and used as aprobe.

5. Chrornosome counts and, determinatiqn of pollen fertility

Root tips were excised from seedlings grown inv i t ro and pretreated wi th 2 mM 8-hydroxyquino-I ine at room temperature for 4 to 5 hr . They were

then f ixed in a solut ion of absolute a lcohol-acet icacid (3:1, v /v) at 4 "C for at least 24 hr , and hy-d ro l yzed w i th 1N HCI a t 60 'C fo r 10 m in . Roo tt ips were then r insed wi th d is t i l led water , s ta inedwi th 2 % aceto-orcein, and observed under a l ightmlcroscope.

To assess pol len fer t i l i ty , mature pol len gra ins

were taken from potted plants grown under nat-ural conditions, stained with 2 o/o aceto-carmine.and observed under a l ight microscope.

Results and Discussion

ln L. regale xL. rubellum, relative length of pol-

len tubes after stigmatic poll ination at anthesiswas 100 7o 96 hr af ter pol l inat ion, and a f ru i tproduct ion f requency of 80.6 7o was obta ined 80days af ter pol l inat ion. Al though the seeds in thesefru i ts looked t ransparent and d id not have anyhardened t issue, 3.3 o/o were observed to conta in

an embryo. The seeds d id not germinate in soi l orin v i t ro on ovule cul ture medium. Embryos f rom l .regalexL. rubel lum normal ly developed in v ivo as

do those from self-poll inated L. regale about 30

days af ter pol l inat ion (F ig. 1A), but thei r develop-ment gradual ly became retarded and stopped 60

days af ter pol l inat ion (F ig. 1B). This observat ionindicates that fer t i l izat ion may occur normal ly butthat a post- fer t i l izat ion barr ier ar ises. To over-

come th is barr ier , ovule cul ture was used on sam-ples col lected at var ious in tervals af ter pol l inat ion

to rescue the in terspeci f ic hybr id embryos (Table

1 ) .

Young ovu les i n ova ry -s l i ce d i sks , 10 to 20

days af ter pol l inat ion, grew s l ight ly even af ter

they were subcul tured on a f resh medium. Using

the ovary-s l ice cul ture technique, seedl ings were

successfu l ly obta ined f rom very young embryosrescued 10 days (N i im i e t a l . , 1995 ) and 20 days(Hayashi et a l . , 1986) af ter pol l inat ion in an in-

t raspeci f ic cross of L. fonnosanwrn; but in ourstudy young embryos of L. regale x L. rubellunt

could not be rescued by th is technique. We at t r ib-ute the fa i lure to the d i f ferences in embryo v igor .A s imi lar resul t was repor ted for in terspeci f ic

crosses among Li l ium species by Okazaki et a l .( 1 9 9 4 ) .

From 5.3 to 6.7 7o of ovar ies cul tured 30 to 60days af ter pol l inat ion y ie lded hybr id seedl ings. Amicroscopic examinat ion of ser ia l sect ions of the

cul tured ovules showed that some embryos con-

t inued thei r normal development 120 days af terpol l inat ion (F ig. 2) . However, seedl ings obta ined

by ovule cul ture 50 and 60 days af ter pol l inat ion

of ten exhib i ted some abnormal i t ies such as cal lus-ing and swel l ing. Therefore, rescuing ovules 30 or40 days af ter pol l inat ion seems opt imum forobtaining normal seedlings in L. regale x L. rubel.l u m .

Table 1. Seedling production from l. regale X L. rubellwmcrosses by ovule culture at various times after poll i-nation'.

1996.

In the rec iprocal cross, l . rubel lum xL. regale,no capsules were obta ined by st igmat ic pol l inat ion

at anthesis . A microscopic observat ion revealed

that growth of. L. regale pollen tubes was inhibited

soon after germination on the stigma of. L. rubellum

; the re lat ive length of pol len tubes was below 10

% even 96 hr af ter pol l inat ion (Table 2) . Thus,uni la tera l incongrui ty (Hogenboom, 1984) was

observed in the rec iprocal crosses between I .

rubellum and l. regale ; it has also been reported

for several in terspeci f ic combinat ions in the genus

Lil ium, including those between L. longiflorum andL. dawr icum (Asano, 1982), L. rubel lum and L.xfor-rnolongi (Okazaki et a l . , 1992), / - x 'Or ienta l ' hyb-

r ids and I . x 'As iat ic ' hybr ids (Okazaki et a l . ,1994), and L. x 'Asiat ic hybr ids ' and l . concolor(Okazaki et a l . , 1995). The b iochemical bases for

these unilateral incongruities in the genus Lil iumneed to be c lar i f ied.

To promote pollen tube growth in L. rwbellwrn xL. regale, two pol l inat ion methods, s t igmat ic andcut-sty le pol l inat ion, were t r ied us ing f lowers of

Table 2. Relative length of pollen tubes and swell ing ovar.ies after stigmatic or cut-style poll ination at varioustimes before or after anthesis in L. rubellum X L. re-gale

RelatiVe length of

p o l l e n t u b e s ( % ) X Swell ingovaries*( % )48 hr after 96 hr after

pol l ination pol l ination

mlltt lttly

+ 4

+ 5- 2

0

+ 5

O a

9.6a

4.Oa

8 5a

26.7b

43.4c

43.lc

44.2c

O a

6.2a

8.2a

12.2a

75。9d

79.8d

88.5e

100.Oe

0

0

0

0

0

0

33.3

33 3

0

0

00

0

6 . 3

6 . 7

5 . 3

6 . 0

ST

十

+

十

2

1

0

1

2

3

CS

Days afterpol l ination 龍憔y盗11

No. of %of ovulesseedlings developing

obtained seedlings

Stigmatic pol l ination was carr ied out at anthesis. Data

were recorded 6 months after pol l ination

SL * OL; ovary-sl ice culture for 1 month fol lowed byovule culture. O: ovule culture

ST, st igmatic pol l ination; CS, cut.style pol l ination0, at anthesis; -, day (s) before anthesis; +, dav (s)after anthesis.Values represent the mean of at least 5 repl icates. Meansin the same column fol lowed by the same letter are notsignif icantly dif ferent (p ( 0.05; Duncan's mult iplerange test).Each pol l ination treatment consisted of 6 repl icates. Datawere recorded 40 days after pol l ination.Not determined

SL+O

SL ttO

O

O

O

O

922

di f ferent ages. The resul ts on the re lat ive length of

pol len tubes and swel l ing ovar ies are summarized

in Table 2; the re lat ive pol len tube length was not

determined af ter cut-s ty le pol l inat ion. In s t igmat ic

pol l inat ion, pol len tube growth was promoted s ig-

n i f icant ly when pol l inat ion was delayed. The re la-

t ive length reached 100 %o 96 hr af ter pol l inat ion

when st igmat ic pol l inat ion was carr ied out 5 days

af ter anthesis . About 33.3 7o of ovar ies became

swol len when st igmat ic pol l inat ion was carr ied out

4 and 5 days af ter anthesis , but these ovar ies

stopped growing 20 to 25 days la ter . Ovar ies

which had been pol l inated 1 and 2 days before

anthesis , at anthesis , and 1 to 3 days af ter anthe-

s is by the st igmat ic method turned brown, wi thout

enlarg ing, soon af terward and eventual ly d ied.

Y N五 mi,M NakanO and K Maki

Thus, inh ib i t ion of pol len tube growth in L.

rwbel lum'xL. regale can be overcome i f f lowers are

pol l inated 4 to 5 days af ter anthesis . Ascher and

Peloquin (1966) repor ted that , in the genus L i l iwm ,

pol len tube growth was promoted when o lder f low-

ers were sel f -pol l inated in sel f - incompat ib le p lants,

t,. tongiflontm 'Ace'. They suggested that the prin-

c ip le const i tuents of se l f - incompat ib i l i ty seemed to

become mi t igated wi th the onset of f lora l senes-

cence. Therefore, an incompat ib le pol len at th is

t ime might escape i ts in f luence. A s imi lar mecha-

nism may a lso be involved in an in terspeci f ic

cross of L. rwbellu'm xL. regale .

On the other hand, cut-s ty le pol l inat ion, which

has been demonstrated to be ef fect ive in overcom-

ing bo th se l f ( L i and N i im i , 1995 ) and c ross - tn -

●

　

ヽ・‘句

「

Fig. 1. Longitudinal sections of 30-(A) and 60 (B) day-old ovaries st igmatic-pol l inated at anthesis in l .

regale x.L rubel lu'm. Bar:0 1 mm

Fig.2. A longitudinal section of a cultured ovule 120 days afLer st igmatic pol l ination at anthesis in l .

regale xL ntbel lum Bar:0.1 mm

FiS. 3. Longitudinal sections of 20 (A) and 40-(B) day-old ovaries st igmatic-pol l inated 5 days after

anthesis in L. rubel l t tm r.L regale. Bar:0.1 mm

”ば　ヽ．●

選甲

J. Japan. Soc. Hbrt Sci .64(4) : 919-925. 1996. 923

compat ib i l i ty (Asano and Myodo, 1977\ , had no

effect on the swell ing of ovaries in L. rubellum xL.regale . Ovaries poll inated at all 3 stages of f lower-

ing by the cut-s ty le method exhib i ted browning

soon af ter pol l inat ion and eventual ly d ied.An examinat ion of ser ia l sect ions of ovules de-

rived from l. rubellum x L. regale which werest igmat ic-pol l inated 5 days af ter anthesis and col -lected 20 days la ter revealed that they had a fewovules, each wi th an embryo (F ig. 3A). However,these embryos grew a l i t t le , developed no endo-

sperm t issue, even 40 days af ter pol l inat ion (F ig.

3B). To rescue embryos, ovary-s l ices were cul -tured for 1 month and the growing ovules excisedfrom the slices were subcultured onto a freshmedium. However, no embryos grew into seedl ingseven 6 months af ter pol l inat ion. Ser ia l sect ionsdisc losed no developing embryos in the ovules cul -tured for BO days. Fur ther exper iments need to beconducted to establ ish an ef f ic ient cu l ture systemfor rescuing young embryos oI L. rubellum xL. re.gale .

Seedlings of L. regale xL. rubellum were exposedto cold for more than 12 weeks to break thei r dor-mancy, and then they were grown in a pot undernatura l condi t ions. To conf i rm thei r hybr id i ty ,nuclear rDNA of some c lones were analyzed. Tota lDNAs of both parents, L. regale and L. rubellum ,and putative hybrids were digested with severalrest r ic t ion enzymes and subjected to analys is .Among the restriction enzymes tested, I l ir dll l wassuper ior to Eco RI and EcoRV for d iscr iminat ingrDNA fragments of both parents (Fig. a). Hin dll l .d igested nuclear DNAs f rom al l putat ive hybr idsconta ined the 23.0 kbp f ragment f rom L. regaleand the 6.4 and 6.1 kbp f ragments f . rom L. rubel -lum , which indicates that these seedlings are in-terspeci f ic hybr ids between L. regale and L. rwbel-lzzn. . Resul ts of chromosome counrs revealed thata l l o f t he hyb r i d p lan ts we re d ip lo id ,2n :2x :24(Fig. 5) . Several hybr id p lants bore l ight p inkf lowers; the color was intermediate between thatof the white L. regale and pink l. rwbellum . Onehybr id c lone produced a double f lower wi th nor-mal s tamens (Fig. 6) . Less than 3 % of the pol len

col lected f rom the hybr ids were fer t i le as deter-mined by aceto-carmine sta in ing.

In the present s tudy, hybr id p lants betweenthese two species were obtained when L. regalewas used as a female parent but none f rom the re-

: : '1 : : t : / t t i ! : : : ; . . : : . , ' r i , : . i . i : : , . : r t i : .

Fig. 4. Blot-hybridization of digoxigenin-labeled rDNA fragments to HindIIIdigested nuclear DNAs of the parentalspecies and their hybrids Arrowheads, from top to bottom, indicate23.0, 6.4 and 6.1 kbp fragments. re.spectively. Lane 1, L. rubel lum; lane 2,L. regale; lanes 3 to 6, interspecifichybrids.

Fig. 5. Chromosomes in a root tip cell ofterspecif ic hybrid (2n: 2x:24\.

an in‐

Y Niimi. M Nakano and K. Ivlaki

Fig. 6. An interspecific hybrid with a doubledf lower.

c iprocal cross. Exper iments should be conducted

in the future to character ize these hybr id p lants

wi th respect to d isease res is tance and envi ronmen'

ta l adaptabi l i ty , and to backcross them to both

parents af ter thei r pol len fer t i l i ty is recovered by

ch romosome doub l i ng (Asano ' 1982 ) '

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摘

リーガルユ リ (Ltιづγπ″gα″)の 強健な性質をヒメ

サユ リ (L.物うι′ιππ)に 導入することを目的として,

両種間で相互交雑 を行った.

1.リ ーガルユ リ×ヒメサユ リにおいては,開 花当

日の柱頭受粉により低率 (33%)な が ら有胚種子が

得 られた.し か し,そ れらの種子はバーミキュライ ト

および試験管内に播種 して も発芽 しなかった。 リーガ

ルユ リ×ヒメサユ リの雑種実生は受粉 30～60日 後に

胚珠培養 を行 うことにより得 られ,そ の頻度は 53～

67%で あった.

2.ヒ メサユ リ×リーガルユ リにおいては,開 花当

日の柱頭受粉では受粉後に花粉管が花柱内で伸長を停

J Japan Soc Hort Sci 64(4):919-925. 1996. 925

t ion of Lit ium: Preliminary results of the applica-

tion of poll ination and embryo-rescue methods.

The L i ly Yearbook of the N. A. L. S. 45 : 28-37.

Van Tuyl , J . M. , T. P. Straathof , R. J . Bino and A. M.

M. Kwakkenbos. 1988. Effect of three poll ination

methods on embryo development and seedset in

intra- and interspecific crosses between seven

Li l ium species. Sex. Plant Reprod. 1 :119-123.

Van Tuyl , J . M. , M. P. Van Crei j , T. C. M. Van Kle in-

wee, J . Franken and R. J . Bino. 1991. Appl icat ion

of in vitro poll ination, ovary culture, ovule culture

and embryo rescue for overcoming incongruity

barriers in interspecif. ic Lil iwm crosses. Plant Sci.

7 4 : 115-126.Wakizuka, T. and T. Nakaj ima. 1975' Development of

proembryo in cultured ovules of. Petunia hybrid.a

Vi lm. Japan. J . Breed. 25 : 16l -167.

胚珠培養によるリーガルユリとヒメサユ リの種 間雑種 の作 出

優 。牧 健 一郎*

新潟市五十嵐二の町 8050

要

上 し,受 精が起こらなかった。 しかし,開 花 2～5日

後に柱頭受粉を行うことにより花粉管伸長がイ足進され,

開花 5日 後の受粉では胚形成が確認された。花柱切断

受粉は胚形成に効果がなかった。開花 5日 後の受粉に

より得られた胚は,胚 珠培養を行っても救出すること

ができなかった.

3.リ ーガルユ リ×ヒメサユリから得られた個体の

雑種性は rDNA分 析により確認された。調査 したす

べての雑種は三倍体であり,花 粉稔性は3%以 下であ

った。雑種4固体の花色は淡桃色であった。また,二 重

咲きの花をもつ雑種も1系統得られた。

新美芳二 ・中野

新潟大学農学部 950■21

*現在 :日本デルモンテllk1 378 沼田市清水町 3748.