Factors Affecting the Regeneration and Growth oI

J. Japan. Soc. Hort. Sci. 54(1) : 82-86. 1985.
Factors Affecting the Regenerationand Growth oI
Bulbletsin Bulb-scaleCulturesof
Lilium rubellum Baker'
Yoshiji NIIvtt
Faculty
Laboratory of Horticultural
of Agriculture,
Niigata
Uniuersity, Niigata 950-21
Science,
SummarY
L i l i u m r u b e l l u m B a k e r b u l b = s c a l e se x c i s e d f r o m b u l b s g r o w i n g i n v i t r o w e r e c u l '
tured at different temperatures, in various lengths of light period, or in a medium
containing different concentrations of a-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA).
Temperature affected the percentage of regenerating bulb-scales, the number of
bulblets per scale, and the growth of bulblets. The optimum temperature for each
of the parameters was 25"C.
The percentage of regenerating bulb-scales and the number of bulbiets per scale
were stimulated in a lighted environment, but the fresh weight of bulblets formed
in this environment was lower than the weight of those formed in darkness.
NAA at concentrations of 0. 05 and 0.1mgll gave a stimulatory effect on each of
BA
the parameters, but the higher concentrations suppressed bulblet formation.
scarcely affected any of the parameters, except in the combination of 0. 1 mgll BA
with 1.0mgll NAA, which enhancedthe fresh weight of the bulblets.
The present study was made to clarLilium.
ify effect of temperature, of length of light
period and of concentration of a-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) on the regenerationand growth
of bulblets in L. rubellum bulb-scale cultures.
Introduction
In aitro asexual propagation has been applied to Litium rubellum Baker for the proliferation of plantlets which were genetically
id.entical to the parent plants (10), because
the extent of rose color, the flowering time,
the length of stem, the number and shape of
As
leaves depend on the parent plants (6).
(
1
1
)
,
t
he
p
a
p
e
r
h
o
w
e
v
e
r
,
p
r
e
v
i
o
u
s
i
n
a
shown
productivity of the explants excised from
each organ oI L. rubellunt parent plants was
{ound to be very low as compared with Z'
Materials and Methods
basal medium consistedof
Medium. -The
Murashige and Skoog's salt (8) together with
2 mglt glycine, 100mg/l myo-inositol, 0.5
mg/l nicotinic acid, 0.5 mg/i pyridoxine-Hcl,
0. 1 mg/l thiamine-Hcl, 50 g/l sucrose, and 7
g/l Dif co Bacto agar. Different concentrations of growth regulators NAA and BA
were added to the basal medium in each experiment, and the concentrationsare shown
in results. All media were adjusted to pH
5.6-5.7with 0.1N NaOH and 0.1N HCI before the addition of sucroseand agar. The
medium was dissolvedand about 20 ml of it
was then poured into each 50 ml Erlenmeyer
flask, all of which were then cappedwith aluminium foil and autoclavedfor 10 minutes at
tongifi.orum (I9). To increase the number of
bulblets for a short period, the bulb-scale culture, in which bulb-scales are excised from
bulbs form ed in aitro, seemed to be one of
s e v e r a l u s e fu l m e t h o d s . H o w e v e r , i t h a s
been reported that in axenic bulb-scale cultures concentrations of growth regulators (1,
1 3 ,1 9 ) a n d c u l t u r e c o n d i t i o n s ( 2 , 7 , 1 9 ) s u i t able for the development of bulblets depend on
I Received for publication February 1, 1985.
82
FACTORS AFFECTING THE REGЁ
NERAT10N AND GROWTH OF BULBLETS
721'C under a pressure of I.2 kg/cm2.
Bulb-scale culture.-Unexpanded
leaves
were dissected from mother plants and cultured f.or I20 days in darkness in a basal medium supplemented with 1.0mgll NAA and
0. 1 mg/l BA, according to the procedures described previously (10, 11). Developing bulblets were dissected and subcultured in the
basal medium at 24-rlC in darkness for a few
months before experiments on bulb-scale cultures began.
All bulb-scales, excluding the innermost
scales near the growing point, were dissected
from
the subculturing bulbs. The bulbscales, weighing more than 10 mg in fresh
weight, were chosen, and 3 to 5 bulb-scales
were placed in each of flasks with the abaxial
side down on the basal medium or on the basal medium supplemented with growth regulators at various concentrations.
Bulb-scales were cultured for 60 days in
,darknessor in a lighted environment consisting of 20 W, white fluorescent tubes, giving
a b o u t 1 0 0 0 - 1 2 0 0l x a t t h e c u l t u r e s u r f a c e .
The number of bulb-scales cultured in each of
the experiments is shown in results.
Determining number and ueight of deaeloped bulblss5.-[1
the end of the incubation period, bulb-scales were rernoved from
each of the flasks for evaluation. After the
number of bulb-scales forming bulblets were
.determined, the average number and fresh
weight of the bulblets formed were evaluated
in each of the flasks. Fresh weight of rooted
bulblets was determined after removal of the
roots. The results were subjected to statis-tical analysis at the 5f level following
Duncan's multiple range test (3).
Results
Efect of temperature. -Table
I shows
the results obtained at the various temperature, 15,20,25 and 30'C. The highest percentage of regenerating bulb-scales was observed at 25oC, and 30'C reduced the regeneration rate with occasional death of bulb,scales. The number of bulblets per scale and
the fresh weight of bulblets were greatest at
25C, and the nearest suboptimum temperaiure for the formation of bulblets was 30oC.
Table 1.
;
83
Regeneration and bulblet growth in bulb*scales
cultured in a basal medium supplemented I.0mg/l
NAA and 0. Lmgll BA, at different temgieratures
in darkness for 60 days, Eighteen bulb-scales were
cultuied
in
columns
not
each of
significantly
the
followed
treatments.
by
the
different at the 5fi
Means in
same letrer
are
level.
Tem3m“
ЩttitS WdSpr tt
Number of
Number Of
NIean fresh
15
66b
1.4b
34b
20
83ab
l.4b
23b
25
89a
2.3a
57a
30
66b
1 8ab
14b
at which their grOwth,hOwever,was extreme=
ly inhibited. n ェ
conclusiOn,
259C was the
Optimum temperature fOr all parameters.
E∬ `ε′響 ιηgιん 9ノ ι
′
gんι夕ιだοノ.‐ Taもlel
2 shOws the rcsults Obtained in variQus
lengths Of light periOd. The percentage Of′
regenerating bulb― scales increased as the,
length Of lightiperiOd increased, althOughl
thё
re were statisticallナ
■O differences amonξ
tre五
ぜ
mёnts.This was alξo true for thき
num・」
ber of bulblets per scale, which was gieatest
i■a continuOusly lighted envirOnment.and
10west in darkness. In cOntFast, the fFcshリ
weight Of bulblets was greatest in idark,essl
althOugh theFe was statistically no differOncel
between thc fresh Weight Of the bulblets
ve10ped in darkness and thOse deve10ped in a
:
cOュtinuOusly lighted envirOnment.
E∬ ιειQ′ N五 五 α′
ο″ιο″ q′ N五 五 」
η θο2‐
ιJηαι
Jθ
η ωゲ
ι
ん B4.言
Table 3 gives the re.
s■
lts Of grOwth regulatOrs.AS COmpared wilh
the cOntr01,the additiOn of grOwth regulatOrs
generally reduced the percentage of regene‐
rating bulb―
scales, particularly at Of
4 5 mg′
Table 2.
RegeneratiOn and bulblet grOwth in bulb―scttles
cultured at variOus lengths Of light period(h)at
24士 rC Thirty bulb― scales in each Of the treat―
ments were cultured in the same kind Of medium
as h Table l. Statistical analysis as in Table l.
Length
of light
period
(h)
Number Of
Number of
NIlean fresh
軒
:ll 撻
紺」
顎
ル S:翼
0
80a
l. o-
8
83a
o
16
93a
24
100a
2.8^b
a A^
qabc
60"
37b
aAb
47'b l
YOSHIJI NHMI
84
Table 3.
Regeneration and bulblet growth in bulb-scales'
cultured in a basal medium supplemented with
various concentrationsof NAA alone or of NAA
together with BA. Forty scales in each of the
treatments vrere cultured at 24+-1T in a contin'
uously iighted environment. Statistical analysis
as in Table 1.
GrOwth regulators Number of
bulb-scales
(mgノ′
)
Number of
bulblets
with bulblets per scale
/ o/\
NAA BA
Mean fresh
weight of
bulblet,
\mg,
88a
2.ObCde
16de
0.01
0
80ab
2.5abcd
23bCdC
0.05
0
88a
3.4a
32bC
0.1
0
83ab
3.Oab
37b
0.5
0
58bCde
l.9bCde
29bCde
1.0
0
30fg
l.Oef
32bC
2.0
0
35CFg
O.9ef
14e
15g
O.2f
20Cde
23bCde
0
30
0(contr。
0
1)
0.001
75abc
2.lbCde
0.01 0.01
75abc
l.8bCde
16de
0.01
73abc
l.6Cde
24bCde
70abcd
2.7abc
25bCde
0 01
0.1
0.1
0.001
88a
3.4a
27bCde
0.1
0.1
90a
2.2bCd
30bCd
1.0
0.001
55Cdef
l.9bCde
22bCde
1.0
0.01
45deFg
l.4de
23bCde
1.0
0.1
65abcd
l.6Cde
55a
0.1
0.01
NAA and more, and at 1.0 mg/l NAA in
combination with BA. The number of bulblets
per scaleincreasedwhen only NAA at the concentrations of 0. 05 and 0. I mgll was added.
The addition of BA with NAA scarcely affected the increasein the number of bulblets,
except for 0.01 mg/l BA together with 0.1
mg// NAA, in which 3.4 bulblets developed
The fresh
as shown in 0.05 mg/l NAA.
weight of bulblets was greatest in 1. 0 mgll
NAA in combination with 0. 1mg/l BA, in
which the number of bulblets per scale rvas
lower than the control. NAA at concentrations from 0.01 up to and including 1.0
mg/l also stimulated the growth of bulblets'
From a comprehensiveanalysis of the combined parameters,NAA at the concentrations
of 0. 05 and 0. L mgll seemed to be most
favorable for the production of bulblets.
Discussion
The present study showed that temperatures affectedall parameters,i. e., the percentage of regeneratingbulb-scales,the number
of bulblets per scale, and the growth of bulb-
lets (Table 1). The optimum temperature for'
the in ailro production of bulblets in other bulb plants was as follows :25C was favorable for bulblet formation in L' speciosum
(2) ; 2I.6 or 24.8"Cfor bulblet growth in hvacinths QD ; and 25'C for all parameters in
narcissus(17). Furthermore, it has been
reported that a temperature increasefrom 15'
to 22oC increasesthe active sucrose uptake
rate about 50fr, in the in 'uitro root discs of
sugar beets (16). These data show that the'
optimum temperatures for in oitro development of buiblets probably lie from 20 to 25CThe range of temperatureshas been not only
applied to the in uiao scale propagation of
Lilium (LB), but also has favored scale initiation and filling of L.longiflorum daughter
bulbs (20). Hence, it could be concludedthat
25'C was the favorable temperature for the ir
aitro propagation of L.rubellum Baker.
Table 2 showed that illumination, particularly continuous light, strongly promotedl
bulblet formation. The effect of illumination on bulblet formation dependedon Lilium : The lighted environment was favorable
'Osnat' (7) ; but unfavoraf.or L. Iongiflorum
'Ace' (19) and L.
ble both f.or L.longifiorum
oriental hybrids (13). Darknesswas also favorable for the regeneration and growth of
bulblets in the leaf segment cultures of' L.
rubellum Baker (10). The discrepancyseems
to be due partly to the differencesin the physiological states of the explants investigated.
Table 2 also showed that the growth of bulblets f ormed in darkness was superior to
those formed in a lighted environment. This
seemsto be related with the number of bulblets formed on a bulb-scale : Since rooting
in developingbulblets began 1 to 1. 5 months
after bulb-scale culture (data not shown),
the developingbulblets seemedto uptake nutrition through each individual bulb-scale
nearly throughout the entire period of bulblet growth ; and as a result, the nutrient
competition in the bulblets grown in Iighted
conditions is assumedto be greater than in
those formed in darkness.
The present study gavd the following
growth-regulator results : NAA was clearly
required for the formation and growth of Z.
FACTORS AFFECTING THE REGENERATION AND GROWTH OF BULBLETS
rubellum bulblets ; and low concentrations
'were more favorable for them. Growth of
isolated bulblets of. L. rubellum Baker was al:so stimulated in the presence of 0. 1 mg/l
NAA or of 0.1mg// NAA in combination
with 0.001mg// BA (12). Similarly, it has
'beenreported elsewherethat the development
,of bulbets was stimulated at 0.03 mg/l NAA
tn L.longiflorum (19) and 0.lmgll in L. spe.ciosum (1). This may be related to the fact
that Lilium bulb-scalesordinarily form new
bulblets in the in aiuo scalepropagation with.out difficulty. The present study also show'ed that BA was not a critical addendum to
increasethe number of bulblets. The same
phenomenahave been observedin severaloth.er bulb plants (4,5,15), and furthermore,
'cytokinin 2 iP suppressed the regeneration
.and growth of bulblets in L.longifl.orum
(19). Hence, cytokinins seem to be less important addendumin the bulb-scalecultures
in Lilium, although Murashige indicated that
cytokinins were one of critical compoundsin
increasing the proliferation rate of herba.ceousand woody plants (9).
7.
10.
Acknowledgements
The author is grateful to Mr. p. J. Runkel
,of the Purdue University for correcting the
English text.
Literature Cited
1. Ae.nrRUr,J. Van and G. J. BLoM-BARNHooRN.
14.
1981.
Growth regulator requirements for adventitious regeneration from Lilium
bilb_
scale tissue in vitro, in relation to duration
of bulb storage and cultivar. Scientia Hortic.
14:267-268.
AARTRIJK,J. VAN ANd G. J. BLOM-BARNHOORN.
1983. Adventitious bud formation from bulb_
scale explants of Lilium speciosum Thunb. in
3.
5
vitro. Z. Pflanzenphysiol. 110 : 3S5-863.
DUNcAN,D. B. 1955. Multiple
range
and
multiple F tests. Biometrics lI : \-24.
HUSSEY,G. 1975. Propagation of hyacinths by
tissue culture. Scientia Hortic. 3 : 2l_2g.
HUSSEY,G. 1975. Totipotency in tissue ex_
plants and callus of some members of the Liliaceae, Iridaceae, and Amaryllidaceae.
J.
26 : 253-262.
IKEDA,Y. 1977. Studies on Lilium rubellum
Baker. 2. Regarding variation and utilization
exp. Bot.
17.
85
as horticultural variety. Bull. Niigata Hort i c . E x p . S t a t . 6 : 4 2 - 7 0 ( I n J a p a n e s ew i t h
English summary).
LESHEM,
B,, H. LILIEM-KIPNIS,and B. STEINITZ.
1982. The effect of light and of explant
orientation on the regeneration and subsequent growth of bulblets on Liliurn longiflorum Thunb. bulbscalesections cultured in
vitro. Scientia Hortic. IZ : 129-186.
MURASHTcE,
T. and F. SKooc. 1962. A revised medium for rapid growth and bioassays
w i t h t o b a c c ot i s s u ec u l t u r e d . p h y s i o l . p l a n t .
15 : 473-497.
MURASHIGE,
T.
1974. Plant propagation
through tissue culture. Ann. Rev. plant
Physiol. 25 : 135-166.
NrrMr,Y. and T. ONozAwA. l97g. In vitro
b u l b l e t f o r m a t i o n f r o m l e a f s e g m e n t so f l i l ies, especially Lilium rubellum Baker. Scientia Hortic. 11 : 379-389.
NrrMr,Y. 1984. Bulblet-productivity of explants from scales, Ieaves, stems and tepals
of Liliurn rubellurn Baker. Scientia Hortic.
22:39L-394.
NIIMI,Y. 1984. Effect of a-naphthaleneacet i c a c i d a n d 6 - b e n z y l a m i n o p u r i n eo n t h e d e velopment of excised-bulbs (Lilium rubellum
Baker) cultured inztitro both in diffusedlight
and in continuous darkness, and the leaf
emergencefrom the bulbs e'zviao. J. Japan.
Soc. Hort. Sci. 53 : 59-65 (In Japanesewith
English summary).
NovAK,F. J. and E. PErRri. 1981. Tissue culture propagation of Lilium hybrids. Scientia
Hortic. 14 : 191-199.
PIERIK,
R, L. M. and M. A. RUIBING.1973. Re.
g e n e r a t i o no f b u l b l e t so n b u l b s c a l es e g m e n t s
of hyacinth in vitro. Neth. J. agric. Sci.
21,i 129-738.
PIERIK,R. L. M.
and
H. H. M. STEEGMANS.
1975. Effect of auxins, cytokinins, gibberelIins, abscisicacid and ethephon on regenerat i o n a n d g r o w t h o f b u l b l e t s o n e x c i s e db u l b
s c a l e s e g m e n t so f h y a c i n t h . P h y s i o l . p l a n t .
34 : 14-17.
R. A., J. Darc and R. E. WySE. 1983.
SAFTNER,
S u c r o s eu p t a k e a n d c o m p a r t m e n t a t i o ni n s u g ar beet tap root tissue. Plant Phvsiol. Z2:
1-6.
SEABRoOK,
J. E. A. and B. G. CUMMTNG.19g2.
In vitro morphogenesisand growth of. Narcis s z s i n r e s p o n s et o t e m p e r a t u r e . S c i e n t i a
Hortic. 16 : 185-190.
SHENK,P. K. and J. BooNrJES.1970. Lilies in
the Netherlands. R. H. S. The lily year
‐r i‐
book. 33:47--57.
19.
・
1 .
YOSHIJI NHMI
WANQ Y.T.and A.N.RoBERTS.1984. Inlu‐
20.
ence of air and soil temperatures on the
zπ ιο″gお
〃′
growth and development of ι
STIMART Do P.and Pa Do AscHER.1978.Tissue
culture of bulb scale sections for asexual pro‐
″g ′
ο
π Thunb. J.
″π
βο
p a g a t i O n o f′
Z ′′
r“
メar“π Thunb. during different grOwth
phase.J.Amer.Soc.Horto Sci.108:810-815.
Amer. Soc. Hort. Sci. 103:182-184.
ヒメサユ リの りん片培養における子球の形成及び生長に及ぼす要因
新 美 芳 二
新潟大学農学部 9 5 0 - 2 1 新潟市五十嵐 2 の 町
摘
試験管内で ヒメサ ユ リの子球 の生産を効果的にす す め
るため,試 験管内で育成 された 子球 の りん片を外植体 と
C)
して用い,そ れ らを異なる培養温度 (15,20,25及
び30°
及び 日長 (0,8,16及 び24時間), あ るいは生長調整物
質 (NAA及 び BA)を 種 々の濃度 で 添加 した培地で培
養 した .そ して,各 条件下 で培養 した りん片の子球 分化
率, りん片当た りの子球数及 び子球生体重を調査 した 。
培養温度は りん片 の子球分化率, りん片 当た りの子球
Cが 子球 の
数 及び子球生体重 の いずれ に も影響 し, 25°
形成及び生長に最適であった 。
要
明条件は子球 の分化率及び子球形成を促進 し, 特 に2 4
時間 日長の連続光は著しい効果を示 した。暗条件では子
球の形成は劣ったが, 形 成された子球 の生体重は最大で
あった。
O . 0 5 及び 0 . l m g ″N A A の 添加は子球の 形成及び生
長を促進 したが, 1 . O m g ′′以上の濃度では 阻害的に作
用 した。N A A と ともに添加した B A は , 0 . l m g ′′B A
′N A A を 添加 した区で形成 された子球の生長
と1 . O m g ノ
を促進 した以外は りん片の子球分化率及び子球形成数に
は とんど効果がないことがわか った.