Plant Science Letters, 13 (1978) 281--285
© Elsevier/North-Holland Scientific Publishers Ltd.
281
REGENERATION OF APPLE PLANTS FROM SHOOT MERISTEM-TIPS
W. DAVID LANE
Agriculture Canada Research Station, Summerland, B.C. VOH I ZO (Canada)
(Received March 16th, 1978)
(Revision received June 6th, 1978)
(Accepted June 6th, 1978)
SUMMARY
Plants of apple (Malus domestica Borkh.) were regenerated from proliferating
meristem-tips grown on nutrient medium. Only benzyladenine (BA), at an
optimum of 5 × 10 -6 M, was required for initial growth and development of
meristem-tip explants which produced proliferating shoot cultures in high
frequency. Naphthaleneacetic acid (NAA) at 10 -s M was used to initiate roots.
Plantlets were then transferred to a growth regulator free medium where
roots developed fully before potting. Temperatures below 28°C and high salt
concentration decreased rooting efficiency.
INTRODUCTION
Meristem-tip culture has recently become an important technique, particularly with vegetatively-propagated species. Very rapid regeneration rates
have been achieved with a number of different species [ 1,2] and the technique
is particularly valuable for rapid clonal multiplication [3]. It is also used for
producing virus-free plants [4 ], international exchange of disease-free plants [5]
as well as such plant breeding functions as germplasm storage [6] and maintaming, in healthy condition, special breeding lines [ 7].
Apples are usually multiplied by grafting the fruiting cultivar onto a rootstock. Regeneration from meristem tips promises to be a quicker and cheaper
method. Self-rooted trees from culture would also avoid graft incompatabilities
and diseases introduced during propagation.
Apple has been cultured previously but shoot multiplication was not achieved
and rooting was either low in frequency or did not occur [8,9]. Jones [10]
reported better results culturing shoot tips (1--2 cm long) of rootstock clones
Abbreviations: BA, benzyladenine; GA3, gibberellic acid; IBA, indole-3-butyric acid; NAA
naph thaleneacetic acid.
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but presented little quantitative data. The present paper describes the procedure for regenerating apple plants from proliferating meristem-tips.
MATERIALS AND METHODS
Malus domestica Borkh. 'McIntosh' seedlings were used in all experiments;
in addition 'Mclntosh' (MacSpur ®) and 'Delicious' (Harrold Red) were often
included. Shoots were collected from both greenhouse and field grown trees,
the outer tissue cut away, and the tip dipped in 70% ethanol (5 s) then blotted
and dissected. Meristem-tips (approx. 0.5 mm wide) containing 3--4 leaf
primordia were carefully dissected from the shoot tip using a scalpel (Brad
Parker No. 11 blade), a Spencer stereo microscope (× 25) and a laminar flow,
sterile air cabinet.
Immediately after dissection tips were placed in 2 × 15 cm glass test-tubes
containing Murashige and Skoog medium [ 11 ] with 2% sucrose plus various
growth regulators. The medium was solidified with Difco Bacto Agar (0.7%)
purified by washing thoroughly in distilled water. Indole-3-butyric acid (IBA),
NAA, BA, and gibberellic acid (GA3) were obtained from Sigma Chemical Co.
Sterilization was by heating to 121°C for 15 min after adjusting the pH to 5.3.
The test-tubes containing the meristem-tips were closed with metal caps,
sealed with Parafilm ® and incubated in a growth chamber illuminated with
cool white fluorescent lamps adjusted for a 16 h light period and 28°C day,
22°C night temperature. Light intensity was 1500 lux at test tube level. Rooting
experiments were in other similar growth chambers with a light period of 18 h
and an intensity of 800 lux.
Experiments investigating initial growth and rooting consisted of 10 replicates, those investigating multiplication, 5. All experiments were repeated at
least twice. Multiplication and rooting experiments were initiated with single
shoots (1--4 cm long) obtained from multiplying cultures.
RESULTS
Initiation and Multiplication
The first transfer to fresh medium was after 6 weeks growth, by which time
a rosette of 6--10 new leaves had developed. T w o months later actively multiplying cultures with 30--50 shoots were well established. Subculturing was
then initiated and repeated at 1 month intervals for 8 months without a decrease in shoot production. Multiplying shoots developed from axillary buds.
Explants from rapidly growing greenhouse trees initially grew more rapidly
than meristem tips from field trees, however, growth rates soon equalized.
Explants from dormant trees grew well, even if chilling requirements had not
been full satisfied, but meristem-tips obtained from larger buds near the branch
terminus grew better than smaller basal buds. The pattern of development of
cultivars was the same, although there were small differences in rates of growth,
in vitro grown shoots resembling those grown in vivo, but lacking trichomes.
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Contamination o f new cultures was less than 5% and 80% of explants were
established as multiplying shoot cultures (Fig. 1 ).
The o p t i m u m c o n c e n t r a t i o n of BA for shoot multiplication was 5 X 10 -6 M
(Fig. 2). If no growth regulators were in the medium, growth slowed and
eventually stopped; at 10 -s M, BA was toxic and the cultures died. Suboptimal
concentrations o f BA resulted in shoot multiplication but at a slower rate.
The influence o f GA3 and NAA in com bi na t i on with 5 × 10 -6 M BA was
also determined (Fig. 3). When GA3 and NAA were combined with BA inhibition occurred, NAA having the greatest effect.
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Fig. 1. A- S h o o t m u l t i p l i c a t i o n in m e d i u m c o n t a i n i n g BA (5 × 1 0 - ' ) . × 1 ; B: R o o t initials
o n s h o o t f r o m m e d i u m c o n t a i n i n g N A A ( 1 0 - 5 M ) ; C: P l a n t l e t w i t h r o o t s w h i c h d e v e l o p e d
a f t e r t r a n s f e r to g r o w t h regulator-free m e d i u m ; D: P l a n t in soil.
284
Root Initiation and Development
Rooting consisted of root initiation and root development phases. NAA at
10 -s M in a medium containing half the normal concentration of salts was
best for induction of root initials. After 4 weeks incubation many root initials
but little callus had formed. Full root development was inhibited by this concentration of NAA so plantlets were transferred to a growth regulator free
medium for 3 weeks.
Temperature during root initiation was very important to rooting success
(Fig. 4), the optimum being 28°C. At lower temperatures shoots first accumulated anthocyanins then became chlorotic. Rooting efficiency also decreased
if IBA was substituted for NAA or if the salt concentration was standard.
Reducing the sucrose concentration below 2% resulted in greener and healthier
appearing shoots but root initiation decreased in proportion to the sucrose
reduction. Sucrose concentrations higher than 5.2% also resulted in a sharp
reduction of rooting.
Rooted plants were transplanted from culture tubes to pots containing sand:
perlite, 1 : 1. Humidity was gradually lowered and the plants then transferred
to the greenhouse.
DISCUSSION
These experiments show the important role of BA in the growth and development of apple meristem-tip cultures. Axillary bud development, as a response
to BA, is known to vary with species and concentration [12], the optimum of
5 × 10 -6 M shown here being similar to that of rose [13] also a w o o d y species
and related to apple. The inhibition of shoot multiplication by NAA in the
present experiments appears similar to previously reported inhibitory effects
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Fig. 2 - - 4 . 2: I n f l u e n c e o f B A c o n c e n t r a t i o n o n m u l t i p l i c a t i o n o f n e w s h o o t s (1 c m or
longer) fromasingleshootafter8weeks;3:InfluenceofBA(5
× 1 0 -6 M ) , G A 3 (5 x 10 -6 M)
a n d N A A (5 × 1 0 -7 M ) o n m u l t i p l i c a t i o n o f n e w s h o o t a n d b u d s f r o m a s i n g l e s h o o t a f t e r 5
w e e k s ; 4: I n f l u e n c e o f t e m p e r a t u r e ( T z = 2 8 ° C : 2 2 ° C , d a y : n i g h t , T 2 = 2 3 ° C : 1 7 ° C , T 3 =
1 8 ° C : 1 2 ° C ) o n a v e r a g e n u m b e r o f r o o t initials f o r m e d p e r s h o o t A--A a n d p e r c e n t a g e o f
s h o o t s w h i c h r o o t e d e - - ~ a f t e r 4 w e e k s in 10- s N A A .
285
o f a u x i n on o t h e r species [ 1,13]. This suggests t h a t a p p l e c u l t u r e s w i t h t h e i r
a b u n d a n c e o f s h o o t tips a p p a r e n t l y have s u f f i c i e n t e n d o g e n o u s a u x i n f o r
g r o w t h ; GAs also a p p e a r s a d e q u a t e as it t o o was slightly i n h i b i t o r y w h e n a d d e d
to the medium.
E x p l a n t s f r o m t e r m i n a l b u d s were established in c u l t u r e with greater freq u e n c y t h a n basal ones. Zieslin [14] has e x a m i n e d t h e e f f e c t o f b u d p o s i t i o n
on g r o w t h r a t e , b y p r u n i n g to d i r e c t b u d g r o w t h , and f o u n d a similar r e s p o n s e ;
basal b u d s grew least a n d g r o w t h increased with successive b u d p o s i t i o n . T h e
smaller size o f m e r i s t e m - t i p e x p l a n t s f r o m basal b u d s c o m p a r e d to t e r m i n a l
ones c o u l d a c c o u n t for their p o o r e r g r o w t h b o t h in vitro a n d in vivo. G r o w t h
o f m e r i s t e m - t i p s f r o m d o r m a n t b u d s w h i c h had n o t satisfied their chilling req u i r e m e n t s was, u n e x p e c t e d l y , n o r m a l . D o r m a n t b u d s n o r m a l l y will n o t g r o w
b e c a u s e o f g r o w t h i n h i b i t o r s and t h e r e s t r i c t e d m o v e m e n t o f o x y g e n and o t h e r
gases to t h e m e r i s t e m [ 1 5 ] . It a p p e a r s t h a t b u d scale r e m o v a l during dissection
a n d t h e free e x c h a n g e o f gases to t h e e x p o s e d m e r i s t e m - t i p s in c u l t u r e released
t h e m f r o m d o r m a n c y allowing g r o w t h .
High a u x i n levels s o m e t i m e i n t e r f e r e with r o o t i n g b e c a u s e excess callus g r o w t h
results [ 1 6 ] . Little callus g r o w t h o c c u r r e d with apple e n a b l i n g t h e use o f a
high a u x i n c o n c e n t r a t i o n t o rapidly i n d u c e r o o t initials. R o o t i n g e f f i c i e n c y was
i m p r o v e d if salt c o n c e n t r a t i o n was r e d u c e d b u t it was also i m p o r t a n t to m a i n t a i n
n o r m a l sucrose levels indicating t h e i m p o r t a n c e o f n u t r i e n t s as well as a u x i n s
for rooting.
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