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INTRODUCTION

Plants of the genus MJusa. the bananas and plantains, are large
herbaceous monocotyledons which grow from massive underground
corms. The transition from vegetative to floral growth and subsequent
fruit formation marks the end of growth for an individual shoot meristem. For all rractical purposes, a given shoot behaves like a monocarpic
plant. The first floral organs to form are the bract-covered hands of
female flowers the ovaries of which will become the fruits. In the dessert
bananas (i.e. thcse clones with sweet fruits which are freauenzlv eaten
iaw) and the solled 'French plantains'. the floral apex can continue to
produce large numbes c f :act -covered male flower clusters until the
fruit bunch is harvested (Barker and Steward, 1962; SimnM,. 196. In
those clones designated as 'F]ie Horn' -'antains (with long horn shaped
fruits generally eaten after cooking). the axis of the male inflorescence
terminates in a large single flower and usually whithers away soon after
all of the fruits have formed (De Langhe. 1961; Tezenas du Mcntcel. De
Langhe and Swennen. 1983). Because the dessert and French plantain
clones have. in theory at least, the morphological capacity for continued
formation of primordia on the flanks of their floral apical meristems, it
seems appropriate to refer to such apices as inetera&na-te. Similarly it is
aoropriate to refer to those floral bud types where it seems an apparent
morphologi Cronauer-Mitra and Krikorian cal impossibility to form
additional bract or leaf primordia and buds on the terminal structure as
determinate. Using the dessert banana clone 'Dwarf Cavendish'as a
prototype. we described the means whereby isolated terminal
indeterminate floral apices could be induced to re-initiate a vegetative
growth mode directly with out the formation of an in.ervening callus
(Crunauer and Krikorian. 1985). In this paper. we report that when zhe
distal segment of the determinate inflorescence of a 'False Horn' plantain
is cultured in acvtokinin containing mediun. adventitious vegetative
shoots can be induced to form in the axils of the bractscars. Once this
occurs. the system behaves and can be manipulated like a vegetative
apex-derived culture.
MATERIALS AND METHODS
Terminal flower buds of three 'False Horn' type plantain clones. 'Harton'.
'Hartor Verde'. and 'Harton Negra' of the 'Harton' type [AAB genome
group according to the convention of Simmonds and Shepherd. (1956)]
were collected in Maracay and Santa Barbara del Zulia,Venezuela as

were corms of Harton Verde' and 'Harton Negra' (of. Haddad and Borges.
1973). Flower buds of the common 'False Horn' plantain known in Costa
Rica as 'Curare' were collected in La Lola, Costa Rica. In the case of the
Venezuelan material, the flower buds were 3 Cronauer-Mitra and
Krikorian carried from the field to a laboratory in Caracas. The majority
of the bracts were removed by hand until the buds measured
approximately 6 to 7 cm in length. They were then surface sterilized in a
2% (vv) comercial bleach (Clorox) solution (final concentration 0.1%
(NaOCl) for 5 min and rinsed four times with sterile distilled water. They
were placed on 10 ml of semisolid medium in 18 mm X 150 mm Kaput
tubes (Bellco Glass. inc.. Vineland. N.J.) which were sealed with Parafilm
and hand carried to Stony Brook. The culture medium was composed of
the mineral salts of Murashige and Skoog (1962) and iron of Singh and
Krikorian (1980). 100 mg L inositol. 1 mg L thiamine HCI, 5 mg/L N 6
-henz'aminourine. 4% sucrose, and 0.7% agar. The pH cf the medium
was adjusted to 5.8 with KOH prior to autoclaving at 121 C and 103 kPa
for 20 min. Upon arrival in Stony Biock. the entire flower buds were
placed :n 50 m! of liquid culture medium of the same composition in 250
ml Erlenmever flasks and placed on a gyrotary shaker at 100 rpm in a
growth chamber maintained at 30 C under a 16:8 L:D cycle (10.2 x 103
lmim 2 Sylvania Grolux wide spectrum). The corms were hand carried to
Stony Brook where vegetative apices were isolated as previously
described (Cronauer and Kri korian. 1984, 1986). The Costa Rican
materi.al was hand carried in an insulated cooler from the field to Stony
Brook where they were prepared as described and placed directly into
liquid medium. Multiplying shoot 4 V Cronauer-Mitra and Krikorian
clusters were removed from the floral axis and transferred to semi-solid
medium in 6.5-cm x 6.5-cm x 10-cm plastic vessels (GA 7 vessels from
Magenta Corp.. Chicago, !L) and were routinely subcultured at 3-4 wk
intervals. Roots were induced to form on individual shoots by placing
them on 10 ml of semi-solid medium containing 1 mg L
naphthaleneacetic acid and 0.025% activated charcoal in Kapuz tubes.
Material isolated for histological examination was fixed in Craf III (Berlyn
and Miksche. 1976), embedded in Paraplast. sectioned at 8 um, and
stained
with an aqueous safranin-fast green series. Material was
Fpepared for scanning electron microscopy by fixing in 3% utaraidehyde
in 0.03M PIPES buffer, pH 8.0, at rcom tEnpeiature for 12 to 24 hr. Fixed
specimens were deh':d:ated In a graded ethanol series on ice, critical
point d:ied. mounted on stubs, sputtered with gold and viewed on an
Amray 1000A scanning electron microscope at 5 Kv.

RESULTS AND DISCUSSION


Flower buds of determinate Musa clones are composed of many layers of
tightly clasping purple bracts and are much more elongated than those
cf indeterminate clones (Fig. 1). When the outermost bracts were
removed, occasionally a "hand" composed of 2 or 3 fruits was
encountered but most of the bracts lacked the expected 5 Jr" CronauerMitra and Krikorian associated nodal cluster of fruits. No nascent flower
promordia were visible to the naked eye (Fig. 2) or in sections. The
removal of the most distal bracts revealed a large terminal flower
structure composed of four stamens and a Dist-il (Figs. 2 and 3). During
the first six weeks in culture, flower buds were transferred to fresh liquid
medium every 7 to .0 days due to the rapid blackening [presumably
phenolic in origin (Palmer. 1963)] of the medium. The remaining bracts
turned brown during this period and were eventuallv removed, thus
revealing the pistil and stamens which eventually blackened and either
atrophied (Fig.4) or were surgically removed. The distal portion of the
peduncle remained green while the cut surfaces which resulted from the
removal of all bracts blackened. Shoot formation began with the
appearance of bud like structures in the axil of the bract scars (Fig.5). As
these buds grew in size. they became green and, in some cases, the
outermost leaf took on the appear ance of a miniature bract in that it did
not com pletely encircle the apical growing point the manner of a
vegetative leaf. However. with subsequent leaf formation. shoot
morphology became indistinguishable from that of shoots originating
from cultured vegetative apices. This response usually occurred at most
of the nodes along the floral axis (Fig. 4). As the tight clusters of shoot
buds expanded. they encircled the Cronauer Mitra and Krikorian floral
axis and later were separated from it with a few scalpel incisions. Overall
shoot morphology at the multiplication stage appeared to be identical to
that of material originating from vegetative apices (Fig. 6). Root
formation proceeded in a similar manner for all shoots regardless of their
origin (vegetative vs. floral) with the first roots appearing 5 days after
shoots were placed on auxin-containing medium. Within three weeks,
the young plantlets were large enough to be transferred to the
greenhouse where plantlets of vegetative and floral origin have been
established. in triolcid (AAA) dessert banana and French plantain (ABE;
clones with floral buds of the type here designated as Indeterminate.
expianted flover buds (order of 2 cm in ength) possessing numerous
hand initials or primordia can be induced to grow and eventually give

rise to adventitious shoot buds. The resulting cultures usually ccn-ain


many diverse floral structures ranging from ovaiies to entire floral buds
several cm in length. Multiplying shoot cultures can be obtained by
repeated selection and subculture of the desired material (Bakry. et al..
1985: Cronauer, (1986). In the case of cultures initiated from primordiafree apical domes. a vegetative growth mode can be induced directly
without the formation of callus or floral structures (Cronauer and
Krikorian, 1985). 70 Cronauer-Mitra and Krikorian The relative ease with
which plantlets could be recovered from indeterminate flcwer buds
prompted us to examine the responsiveness of determinate flower buds
of false horn plantain clones since the tissues of these structures are
fully committed. i.e. there are no floral or vegetative primordia rresent.
In all four clones tested, it was possible tc induce vegetative bud
formation and rowth without the concomitant production of floral
structures even though these vegetative buds formed at the putative
site of a floral organ.The site of origin of these shoot cultures implied an
adventitious origin and this was confirmed by histological examination of
peduncle segments from noncultured flcwei buds as floral primordia
were absent from th*-:ract axais. Examination of cultured peduncle
tissue showed adventitious bud formation (Fig. 7) and subsecuen: A:owth
(Fig. 8). The production of shoot buds at this anomalous locaticn
indicated that the cells in this area were responsive to treatment with an
appropriate growth regulator. in this case. a cvtokinin. Previous reports
on the culture of flower stalk segments in monocotyledonous species
lave deal withe recovery of plantlets from tre existing buds in the orchids
Dendrobium and Phaluenors s (Arditti, 1977). as well as the de nc(,o
formation cf vegetative buds in N rcis~u- (Seabrook, CumminS_. and
Dionne. 1976) and Ho._stf (Zilis and Zwagerman. 1979). and bulblet for 8
Cronauer-Mitra and Krikorian mation in Hyacinthus (Paek. 1982). In
commercial plantations. clonal multiplication of Mus species is
sometimes induced by stripping away the outer leaf sheaths of the
pseudostem and inducing the otherwise dormant leaf-opposed buds to
emerge (Barker. 1959). Presumably, in vitro multiplication of shoot
cultures proceeds in a simiLar manner with the use of high levels of
cytokinin (order of 5 mg'L) inducing pre-initiated bud growth (Dore
Swamy. et al..1983: Cronauer and Krikorian. 1984: Vuylsteke and De
Langhe. 1985: Wong. 1986). However, adventitious shoot formation can
also occur. Histological sections (Fig.8, and scanning electron
micrographs (Fig. 9) have revealed that the site of the vegetative
meristem of a single cultured shoot can contain multiple apices in close

association with each other (Banerjee. Vuylsteke. and De Langhe. 1985:


Cronauer. 1986: Cronauer and Krikorian. 1987). The implications of
adventitious shoot formation on the performance and clonal fidelity
stability of this cultured plant material in the field has yet to be
assessed. ACK1NOWLEDGEMENTS This work was supported by a grant
(DPE5542G55402160) from the United States Agency for International
Development. The help of Professors Carlos Nava and Luis Sosa. Facultad
de Agronomia. Universidad del Zulia, Maracaibo. Venezuela and
Professor 9 Cronauer-Mitra and Krikorian Ludwig E. Muller, CATIE,
Centro Agronomico Tropical de Investigacion y Ensenanza, Turrialba,
Costa Rica in obtaining material is (ratefully acknowledged. We also
thank Dr. Myron C. Ledbetter and Mr. David L. Smith for their help with
the microscopy. 10
Cronauer-Mitra and Krikorian Fig. 1-6. Some
representative stages in the initiation and culture of vegetative shoots
and subsequent plantlet production from floral buds of plantains of
determinate floral type. 1. Terminal floral bud removed from the distal
end of a 'Harton' fruit bunch. X 0.38. 2. The same bud with bracts
removed to reveal the peduncle and terminal floral structure. x 0.48. 3.
Close-up of a 'Harton Verde' floral structure. The four stamens surround
the shorter, bent pistil. x 1.8. 4. Formation of vegetative 'Harton Negra'
shoot buds in the axils of the bract scars along the peduncle. Note the
withered terminal flower structures (arrow). X 1.7. 5. Vegetative bud
formation at the distal end of a 'Curare' peduncle segment. The removal
of the terminal floral structures has left a large, prominent scar(sc).
br=bract scar, b=vegetative bud. sh=shoot. x3.6. 6. Shoot cluster
derived from cultured 'Harton Verde' floral bud. x 1.3. 1 Cronauer -Mitra
and Krikorian Fig. 7-10. Adventitious shoot formation in cultured
vegetative and floral tissues of plantains. 7. Longitudinal section
showing the formation of an adventitious 'Curare' bud in the axil of a
bract. x 90. 8.Later stage in adventitious shoot growth in the clone
'Curare' x 75. 9. Slightly oblique longitudinal section of a 'Cardaba'
(ABB) shoot derived from a cultured vegetative shoot tip which shows
the formation of multiple shoot primordia. x 56. 10. Scanning electron
micrograph of 'Cardaba' material similar to that in Fig. 9. Note that the
outermost leaf primordium surrounds both shoot primordia. x 87.