Plant Science 151 2000 47 – 57
Adventitious shoot mass production of hop Humulus lupulus L. var. Eroica in liquid medium from organogenic nodule cultures
Dora Batista , Lia Ascensa˜o, M. Joa˜o Sousa, M. Salome´ Pais
Departamento de Biologia Vegetal, Centro de Biotecnologia Vegetal, Faculdade de Cieˆncias de Lisboa, Bloco C
2
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1
° piso, Campo Grande,
1749
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Lisboa, Portugal Received 6 May 1999; received in revised form 27 September 1999; accepted 27 September 1999
Abstract
Two efficient methods with high potential for automation are described for mass propagation of Humulus lupulus L. var. Eroica in liquid medium. Suspensions were initiated by culturing petioles directly in a Murashige and Skoog’s modified liquid medium
MS containing 0.8 mgl indole-3-acetic acid IAA and 0.02 mgl kinetin or by inoculating petiole-derived callus into MS liquid medium supplemented with 0.01 mgl IAA or indole-3-butyric acid IBA and 1 mgl 6-benzylaminopurine BAP. In the last case,
transfer of calli to liquid medium proved essential for inducing morphogenesis. Organogenic nodules forming green organogenic nodular clusters GONCs were the basic regenerative structures producing large amounts of adventitious shoots. Both methods
induced the formation of GONCs within 2 – 3 months. Establishment of GONCs cultures in hormone-free liquid medium favoured nodule multiplication and continuous shoot emergence and development. However, GONCs should only be separated from the
suspensions when reaching a high level of differentiation. Histological studies revealed that nodules arise through a complex process that includes four developmental stages: 1 organization centers derived from neoformed xylemic cells; 2 independent
nodules displaying a consistent internal celltissue differentiation with at least three cell types meristematic cells, parenchymatous cells and vascular elements and two cell regions epidermal and cortexvascular; 3 ‘polycenter’ nodules; and 4 organogenic
nodules. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Hop; Humulus lupulus L.; Nodule culture; Liquid medium; Organogenesis www.elsevier.comlocateplantsci
1. Introduction
Hop cultivation is a valuable economic resource in many countries, recognized for at least 10 cen-
turies due to its importance to the brewing indus- try. This dioecious, perennial, climbing plant of
the Cannabinaceae family is exclusively grown for its female inflorescences. The interest of the female
flowers lies in the lupulin glands which produce resins and essential oils that provide bitterness,
flavour and aroma to beer and ales.
From the earliest recognition of the advanta- geous application of hops in brewing, there must
have been a continuing process of selecting im- proved hop varieties [1]. Breeding programmes
were initiated in the beginning of century in UK, United States, Denmark and ex-Czechoslovakia
and have been directed primarily at producing superior bitter or aromatic types combined with
resistance to the major pests and diseases [1,2]. Gene transfer technologies through tissue culture
are an attractive alternative to normal breeding methods since they offer ways of introducing spe-
cific desirable characteristics from several different origins in established hop varieties, without alter-
ing their quality profiles. However, efficient in vitro regeneration of plants is required for the
application
of molecular
genetics to
crop improvement.
Despite the great interest in hops, only a limited amount of work concerning successful plant regen-
eration has been reported, in which regeneration is
Corresponding author. Tel.: + 351-1-750-0165; fax: + 351-1-757- 3625.
E-mail address
:
dorabfc.ul.pt D. Batista 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
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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
