Plant Cell Reports (1988) 7:535-537
Plant Cell
Production of pineapple plants in vitro
M.G. DeWald, G.A. Moore, W.B. Sherman, and M.H. Evans
Fruit Crops Department, IFAS, University of Florida, Gainesville, FL 32611, USA
Received February 19, 1988/Revised version received October 11, 1988 - Communicated by G.C. Phillips
Abstract
In v i t r o culture of pineapple (Ananas comosus)
was studied to determine the e f f i c i e n c y of a x i l l a r y
bud culture f o r rapid propagation of several
c u l t i v a r s . The technique used maximizes the success
rate of various steps in the production of pineapple
plants. Rapid mass m u l t i p l i c a t i o n of p l a n t l e t s
started 9 months a f t e r explanting with a s i g n i f i c a n t
log phase. The number of p l a n t l e t s obtained from the
culture of a single bud by the t h i r t e e n t h month ranged
from 210 to 380 f o r ' P e r o l e r a ' ; 300 to 350 for 'PR-1-
67'; and 40 to 85 for 'Smooth Cayenne'. The method
permits culture of a range of pineapple c u l t i v a r s .
L i t t l e morphological v a r i a t i o n was observed in young
regenerated plants.
Introduction
Pineapple is r o u t i n e l y propagated v e g e t a t i v e l y by
means of l a t e r a l shoots, basal suckers, or crowns.
Plant material is often l i m i t e d , e s p e c i a l l y f o r the
80,000 plants/ha needed in the establishment of new
plantations, and for the propagation of improved
c u l t i v a r s or newly discovered sports.
In v i t r o propagation of pineapple was f i r s t
achieved in 1960 by Aghion and Beauchesne (1960), but
i t was not u n t i l the reports of Lakshmi Sita et a l .
(1974), Teo (1974) and Mapes (1973) that in v i t r o
production of pineapple was considered as a
commercial a l t e r n a t i v e . Most recent reports
(Pannetier and Lanaud 1976, Wakasa et al. 1978,
Mathews and Rangan 1979, Drew 1980, Zepeda and Sagawa
1981) deal only with the factors involved in the
establishment of a x i l l a r y buds in culture. They do
not consider the number of plants regenerated or the
e f f e c t of genotype on culture establishment.
The objectives of the present study were to test
the technique of a x i l l a r y bud culture f o r rapid
pineapple propagation using d i f f e r e n t c u l t i v a r s and to
determine the e f f i c i e n c y of p l a n t l e t production on a
per bud basis. These data would provide an
estimation of explant number, space and time
requirements to meet production needs.
Materials and Methods
To test the f e a s i b i l i t y of a x i l l a r y bud culture
of the various c u l t i v a r s of pineapple, Ananas comosus
(L.) Merr., 20 buds from each of the f o l - i o w i n g - -
c u l t i v a r s were explanted: 'Red Spanish', 'Cambray',
Offprint requests to: M.G. DeWald
Reports
© Springer-Verlag 1988
'Abaka', 'Valera A m a r i l l a ' , 'Bumanguesa',
'Brecheche', 'Esmeralda', 'PR-1-67', 'Perolera' and
'Smooth Cayenne'. Because of the potential for
large numbers of p l a n t l e t s to be produced from a
single bud, 3 buds representative of each of 3
c u l t i v a r s were selected and followed through 13
months of subculture to determine p l a n t l e t
production on a per bud basis. These c u l t i v a r s
were 'PR-I-67', a spiny commercial c u l t i v a r grown
for fresh f r u i t and canning in Puerto Rico;
'Perolera', a smooth-leaved c u l t i v a r grown in the
Northern Andes of South America and used f o r fresh
f r u i t ; and 'Smooth Cayenne', the most widely grown
pineapple c u l t i v a r in the world.
Crowns and stems were rinsed in water,
d e f o l i a t e d and s u r f a c e - s t e r i l i z e d by a g i t a t i o n in a
20% clorox solution (1% sodium hypochlorite) with
2-3 drops of a surfactant (Tween 20) f o r 20 min,
followed by 3 rinses of 10 min each in s t e r i l e
water. Terminal and a x i l l a r y buds were then excised
a s e p t i c a l l y and s u r f a c e - s t e r i l i z e d in 2% clorox
solution for 10 min followed by three 10 min rinses
in s t e r i l e water. Buds were explanted in 60 x 15 mm
petri dishes or subcultured into test tubes
containing Murashige and Skoog (1962) medium,
supplemented with 3% sucrose, 0.8% Difco Bacto-agar,
0.57 mM i n o s i t o l , 1.2 pM thiamine HCI, 10.8 pM NAA and
8.8 ~M BA, adjusted to pH 5.7 and autoclaved at 121 °
C, and 1.1 kg cm-2 for 20 min. Cultures were
incubated at room temperature (24-27 ° C) with a 16 h
photoperiod of 76 Nmol s- I m-2 provided by cool-whit~
fluorescent lamps. Explants were subcultured onto
fresh medium in test tubes at 6 week i n t e r v a l s u n t i l
adequate growth had occurred f o r transfer to l i q u i d
medium.
P r o l i f e r a t i n g explants were m u l t i p l i e d in 50 ml
l i q u i d cultures of the same medium in 125 ml
Erlenmeyer flasks, maintained at 100 rpm on rotary
shakers. Liquid shake cultures were subcultured at
approximately 4 week i n t e r v a l s , when flasks had
become t i g h t l y packed with p l a n t l e t s . In this way,
numerous flasks of callus and regenerating p l a n t l e t s
were obtained from one o r i g i n a l bud.
After each subculture, only p l a n t l e t s 2.5 cm or
larger were harvested. They were transferred to
individual pots or to f l a t s containing a commercial
s o i l mixture and enclosed in p l a s t i c bags, creating
a greenhouse e f f e c t . The plants were incubated in a
growth chamber at 28° C under fluorescent lamps and
gradually hardened o f f by removing the p l a s t i c
covers. Acclimated plants were transplanted to
larger pots and placed in a greenhouse under a black
536

Table I. Mean in v i t r o pineapple p l a n t l e t production per i n i t i a t e d bud.

Mean number of p l a n t l e t s harvested per flask a

Perolera PR-1-67 PR-1-67 Smooth Cayenne

Month
(Subculture) b Bud i Bud 2 Bud 3 Bud I Bud 2 Bud 3 Bud 1 Bud 2 Bud 3 Bud I Bud 2 Bud 3

9 ( 7) 2.5 1.0 2.3 1.5 1.7 1.5 0.0 1.7 0.0 0.5 0.0 4.0
10 ( 8) 2.4 3.3 3.1 3.3 3.5 2.5 3.7 3.8 3.0 0.5 0.5 6.0
11 ( g) 3.7 2.8 3.4 3.1 2.7 3.2 3.2 3.2 2.7 2.0 1.3 2.5
12 (10) 6.6 7.5 5.6 5.0 1.0 c 4.1 6.1 3.8 3.8 3.2 4.8 4.0
13 (11) 12.8 14.8 14.4 11.2 10.5 9.9 6.2 13.8 13.8 4.0 3.8 4.8

aplantlets were larger than 2.5 cm.

bNumber of months a f t e r culture i n i t i a t i o n (number of subculturing a f t e r i n o c u l a t i o n ) .
CSeveral flasks became contaminated during the experiment.

cloth screen to prevent scorching. The screen was

removed 3 weeks l a t e r and surviving plants were
counted. F e r t i l i z a t i o n was i n i t i a l l y done biweekly
with an acid-forming plant f e r t i l i z e r and, l a t e r ,
with a solution of 10% MS salts to remedy apparent
nutrient deficiencies.
The e f f e c t of subculturing into basal medium
lacking phytohormones was evaluated by inoculating 10
g of an a c t i v e l y d i v i d i n g 'Perolera' culture into
flasks containing 50 ml of basal medium with hormones
(10.8 ~M NAA and 8.8 ~M BA) or without. The two
treatments were replicated 9 times.

Results and Discussion

Terminal and a x i l l a r y buds were excised and

placed on solid medium containing auxin and cytokinin
to i n i t i a t e organogenic cultures (Fig. IA). Multiple
buds were i n i t i a t e d from the cultured buds a f t e r 2 to
3 months (Fig. 1B), and the cultures could then be
transferred to l i q u i d medium to enhance
proliferation.
P r o l i f e r a t i n g cultures were obtained from 75% of
the buds explanted, and p l a n t l e t s were regenerated
from a l l I0 c u l t i v a r s tested. No p l a n t l e t s were
harvested from cultures during the f i r s t 9 months;
instead a l l p r o l i f e r a t i n g material was used to
increase culture numbers. Cultures less than 6 months
old usually consisted of a mass of short (<2 cm in
length), thin shoots. However, by 9 months a f t e r
i n i t i a t i o n cultures exhibited a less compact growth
habit and consisted p r i m a r i l y of shoots longer than 2
cm with numerous smaller shoots around t h e i r base
(Fig. IC). Studies done using 3 buds from each of 2
plants of 'PR-I-67' and one plant of 'Perolera' and
'Smooth Cayenne' showed that the total number of
harvestable p l a n t l e t s doubled with each monthly
subculture a f t e r the 11th month (Table I ) . Numbers of
p l a n t l e t s that could be obtained by the 13th month
from a single bud culture varied from 210 to 380 for
' P e r o l e r a ' ; 300 to 350 for 'PR-I-67'; and 40 to 85 for
'Smooth Cayenne' (Table 2). Approximately 25
p l a n t l e t s larger than 2.5 cm could be harvested per
125 ml flask at each additional subculture.
This in v i t r o propagation system was suitable
for a l l c u l t i v a r s tested. However, in the studies
following individual buds, differences were noted Figure I. A x i l l a r y bud culture of pineapple.
between genotypes. 'Smooth Cayenne' was less A. Buds a s e p t i c a l l y removed. B. P r o l i f e r a t i n g
responsive in v i t r o than the other 2 c u l t i v a r s , culture 4 months a f t e r inoculation of bud.
producing fewer p l a n t l e t s per subculture (Table I ) , C. A c t i v e l y d i v i d i n g culture ready for subculture.
and so fewer total plants per i n i t i a t e d bud (Table D. Regenerated plants.
2).