achieved on solid medium via organogenesis. Motegi [3] obtained adventitious shoots from
callus cultures derived from stem explants of Shinshuwase and Italy-2 cvs. Connell and Heale
[4] and Heale et al. [5] developed a green callus system for the regeneration of several cultivars
including Challenger, Eastwell Golding and Ear- lybird Golding. Rakousky and Matousek [6] ob-
tained
direct regeneration
from petioles
or internodes of two Czech commercial clones.
Batista et al. [7] regenerated plants of a sponta- neous Portuguese hop clone and of hop var.
Brewer’s Gold from nodular green callus cultures derived from stem and petiole explants.
For most species cultured in vitro, shoot re- generation is almost always achieved on solid
medium [8]. Adventitious shoot formation and development in liquid medium is not commonly
reported [9 – 13], and it may present several prob- lems, mainly shoot morphological aberrations
and vitrification. Any successful attempt at re- generating shoots in liquid medium is valuable
because it provides an ideal regeneration system. Under these conditions mass production, devel-
opment control and automation are possible, which, together with the concomitant saving in
manual labor and costs, increases the system fea- sibility for commercial large-scale production in
bioreactors [12].
In poplar, plantorgan regeneration in liquid culture was obtained from highly meristematic
nodular clusters termed nodules [14]. Nodules were described as independent, spherical, dense
cell clusters which form a cohesive unit and dis- play a consistent internal celltissue differentia-
tion pattern and a high regenerative capacity [14]. Successful nodule cultures with high regen-
eration potential were also described for Pinus radiata [15], Eucalyptus grandis [16], Chicorium
intybus [17], Nerine sp. [12] and Ananas comosus [18].
This paper reports on the establishment of a highly productive hop regeneration system based
on nodule culture in liquid medium. Two effi- cient induction methods leading to the develop-
ment of organogenic nodules are described for mass production of adventitious shoots of Hu-
mulus lupulus var. Eroica. The histological char- acterization of nodules and their development
are also presented.
2. Materials and methods
2
.
1
. Plant material and suspension cultures establishment
Petiole segments 6 – 8 mm were removed from micropropagated female plants of H. lupulus var.
Eroica, established under sterile conditions as described earlier [7], 3 months after subculture, and
used as the primary explants. Suspension cultures were established in a Murashige and Skoog medium
[19]
MS, modified by Batista et al. [7], supplemented with 15 gl sucrose and 30 mgl
cysteine pH 5.7, according to two different methods: 1 inoculation on solid medium for callus
induction followed by transfer to liquid medium mixed culture; and 2 direct inoculation into
liquid medium liquid culture. All complete culture media were autoclaved at 121°C for 15 min.
Suspension cultures were placed on a gyratory shaker 24 9 2°C at 80 – 85 rpm, under a 168 h
photoperiod with light 73 mEm
2
per s supplied by cool white daylight fluorescent lamps Phillips TLD
18W33. The influence of different parameters on morphogenesis induction was evaluated for each
case. Each experiment was performed using 6 flasks per treatment.
2
.
2
. Nodule induction
2
.
2
.
1
. Mixed culture Callus induction was performed on solid MS
medium 8 gl agar supplemented with 0.01 mgl indole-3-acetic acid IAA or indole-3-butyric acid
IBA and 1 mgl 6-benzylaminopurine BAP, in 9 × 4.5-cm flasks 40 ml-8 petiolesflask. Solid
cultures were maintained at 22 9 2°C under a 168 h photoperiod 33 mEm
2
per s. After 5 weeks, calli were transferred to liquid medium of identical
composition supplemented with 10 mM 1.4615 gl
L
-glutamine. Different inoculum densities were tested using approximately 1, 1.5, 2, 3, 4 g of calli
per 25 ml of liquid medium contained in 100-ml Erlenmeyer flasks. All flasks were closed tightly with
aluminum foil. Callus suspensions were maintained in the same medium for periods of 4, 5 and 6 weeks.
The use of regular subculture intervals of 4, 5, and 6 weeks was compared with subculturing every four
weeks after an initial subculture of six weeks. Whenever green organogenic nodular clusters
GONCs were detectable 8 – 10 weeks, they
were isolated and cultured separately in MS liq- uid medium of identical composition depleted of
growth regulators for shoot emergence and devel- opment. From this point on, both callus and
GONCs cultures were routinely subcultured in this medium every 4 weeks.
2
.
2
.
2
. Liquid culture Liquid cultures were established by inoculating
8 – 10 petioles into 250-ml Erlenmeyer flasks con- taining 50 ml of MS medium with 0.8 mgl IAA
and 0.02 mgl kinetin. Different concentrations of glucose 20, 30, 40 gl and sucrose 20, 25, 30, 40
gl were tested. After callus andor explant mor- phogenic competence induction in this medium
3 – 4 weeks, which was considered the first cul- ture phase, cultures were sequentially transferred,
every 4 weeks, for up to three more culture phases in order to induce nodule formation. Cul-
ture phases differed only in the hormonal supple- mentation of the medium that was as follows:
2nd culture phase 0.1 mgl IAA + 2 mgl BAP, 3rd 0.1 mgl BAP and 4th absence of growth
regulators. In the second culture phase, sugar concentrations above 30 gl were reduced to 20
gl. From the third culture phase onwards, all sucrose and glucose concentrations were replaced
by 15 gl sucrose control conditions. Subcultur- ing from the last culture phase was done every 4
weeks using the same medium hormone-free MS liquid medium + 15 gl sucrose. GONCs, usually
detected between the third and fourth culture phase, were cultured as previously described for
mixed cultures.
2
.
3
. Shoot regeneration GONCs produced by both methods were iden-
tical and thus handled in the same way. Given the continuous production of GONCs by the original
suspensions, new GONCs cultures were repeat- edly established from a single replica. Regener-
ated plantlets with approximately 3 cm tall were excised from the nodular clusters surface and
transferred to micropropagation medium [7]. Af- ter the first production cycle, ranging from the
emergence of the first shoot through to the best moment for plantlet removal 2 – 3 months,
GONCs were used for other production cycles 1 – 2 months each. Based on the high and pro-
longed regeneration capacity of GONCs, this pro- cedure could be repeated at least five times.
Experiments providing the best conditions for re- generation, for both mixed and liquid culture,
were repeated three times using 12 replicates. The efficiency of the regeneration process was evalu-
ated based on the regenerative capacity of GONCs, shoot regeneration rates total and per
production cycle and average number of regener- ated shoots per GONC. Final data correspond to
combined means from the three independent tri- als.
2
.
4
. Plant transfer to soil After development and rooting on micropropa-
gation medium [7] for at least 2 months, plants obtained from both regeneration methods were
potted and hardened in a growth chamber hu- midity progressively reduced to 70, 22 9 2°C,
168 h photoperiod, 60 mEm
2
per s over a 2 – 3- week period.
2
.
5
. Histological study Given the similarity of the regeneration path-
way induced by both methods, histological analy- sis of the initiation and development of nodules
and shoots was performed using only mixed cul- ture-derived samples. Tissues were periodically re-
moved from culture and fixed in formalin – glacial acetic acid – ethanol 70 1:1:18 vv FAA for at
least 3 days at 4°C or freshly sectioned at 9 30
m m with a Reichert freezing microtome. Sections
were stained with iodine greenGrenacher’s alum carmin [20] or directly observed on a Leitz Wet-
zlar Dialux light microscope.
3. Results and discussion
