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0 Centro de Biotecnologia Vegetal, Faculdade de Ciências, Univ. Lisboa, Bloco C2, Piso 1, Campo Grande, 1780 Lisboa, Portugal
Received for publication June 8, 1999. Accepted for publication October 7, 1999.
ABSTRACT
The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.
Key Words: Cannabinaceae • histological studies • Humulus lupulus var. Nugget • internodes • nodules • organogenesis • starch
The totipotent character of plant cells and tissues can be expressed by their ability to regenerate into plants via embryogenesis or organogenesis. Both processes lead to in vitro regeneration and are a major prerequesite for genetic transformation. However, the widespread application of gene transfer techniques for crop improvement cannot be successfully achieved if the processes leading to morphogenesis are not well understood.
Histological, histochemical, and ultrastructural aspects of somatic embryogenesis, whether occurring with or without a callus phase, have been extensively reported (Ho and Vasil, 1983
; Williams and Maheshwaran, 1986
; Stamp, 1987
; Pedroso and Pais, 1993
; Brisibe et al., 1993
; Sagare, Suhasini, and Krishnamurthy, 1995
). A system of efficient regeneration parallel to somatic embryogenesis has been recently proposed (Aitken-Christie, Singh, and Davies, 1988
; McCown et al., 1988
; Warrag, Lesney, and Rockwood, 1991
; Teng, 1997
) and is based on the formation of organogenic nodules that arise directly from the explants or from callus and cell suspension cultures.
Morphogenesis requires an external source of carbon, which is usually supplied in the medium mainly as sucrose. Thorpe and Murashige (1970)
and Mangat, Pelekis, and Cassels (1990)
have shown that in tobacco callus and in Begonia rex stem explants there is a strong accumulation of starch in areas that eventually give rise to meristemoids and shoot primordia in tissues grown on shoot-forming media. High amounts of starch have also been observed during somatic embryogenesis in sugar cane and cassava (Ho and Vasil, 1983
; Stamp, 1987
). The possible role of starch in these processes is not clear. It has been suggested that starch may function as an energy source or may provide osmotica in the form of free soluble sugars (Thorpe, 1980
; Stamp, 1987
). Regardless of its function, starch accumulation/mobilization cycles appear to be a characteristic feature of cells involved in plant morphogenesis.
This paper reports on the histological events leading to nodule formation and shoot regeneration from internode-derived nodules of Humulus lupulus var. Nugget and on the evidence of a starch accumulation/mobilization cycle during this morphogenic process.
MATERIALS AND METHODS
Tissue culture
Internodal explants were taken from 3–4 mo old plants micropropagated in Adams (1975)
media. Explants were first cut into pieces 6–9 mm long and then inoculated in culture flasks (9 x 8 cm) containing ~40 mL of MS solid medium (Murashige and Skoog, 1962
) supplemented with 2 mg/L BAP, 0.05 mg/L IAA, 18 g/L sucrose, and 7.8 g/L agar (Vaz Pereira, Lisbon, Portugal). Control explants were cultured as described above but on medium without growth regulators. The pH was adjusted to 5.7–5.8 before autoclaving at 121°C and 1.01 x 10-5 Pa for 18 min. All cultures were incubated at 25° ± 2°C with a 16-h photoperiod (35 µ mol photons·m-2·s-1) provided by cool-white Philips fluorescent tubes.
Histology and histochemistry
Cultured explants were removed at random at 0, 2, 5, 7, 9, 12, 15, 19, 23, 28, 31, 35, and 45 d and fixed in 30% formalin:glacial acetic acid:70% ethanol (1:1:18) for 72 h at 4°C. Control explants were removed after 8, 15, 28, and 45 d in culture on MS medium without growth regulators and fixed as described above. Sections (20–40 µm) of the fixed internodal segments were obtained using a Reichert freeze microtome and stained for histological and histochemical observations.
For histological studies sections were immersed for 40–60 min in sodium hypochlorite, washed in distilled water, and then stained with 1% Iodine Green for 10 s. Following immersion in acetic acid (1% in distilled water) for 2 min, sections were stained with 1% Grenacher Carmin for 15–20 min. For starch detection, sections were immersed for 20–30 min in sodium hypochlorite, washed in distilled water, and stained with 5% (v/v) lugol solution (Merck, Darmstadt, Germany) (Jensen, 1962
). Sections were then floated on a drop of water placed on glass slides and examined under a light microscope (Leitz-Wetzlar, Germany).
Sections were also taken from cultured explants and embedded in paraffin wax. These explants were fixed as previously described, dehydrated in a graded ethanol series, and then left overnight in 100% ethanol. Material was then passed through graded ethanol/xylene mixtures (100% ethanol; 3:1; 1:1, 1:3, and 100% xylene) and embedded in paraffin wax (melting point: 51°–53°C). Serial sections of 8–12 µm thickness were obtained with a rotary microtome (Reichert Jung, 2050 Supercut, Heidelberg, Germany). Sections were stretched on glass slides previously treated with 100 mg/mL poly-L-Lysine (Sigma), exposed to xylene-ethanol series to remove paraffin and stained with 0.13% methylene blue and 0.1% safranin. They were then mounted in Permount mountant (Fisher, New Jersey, USA).
Scanning electron microscopy (SEM)
Internodes cultured for 45 d, both on organogenesis-inducing and basal media, were harvested and immediately fixed as described above. Following dehydration via a graded acetone series, samples were dried by critical point drying method and sectioned before mounting. Then, samples were sputtered with a thin layer of gold and observed in a Jeol JSM- T220 SEM at 15 kV. Micrographs were taken with a Plus-X-Pan Kodak film.
RESULTS
Transverse sections of internodal explants at day 0 showed a typical dicotyledon stem structure. No starch was detected (Fig. 1). Five days after culture on induction medium, cambial cells started dividing tangentially, leading to an enlargement of the explant (Fig. 2). This enlargement was evenly visible along the explant tissue, so that the explant kept a cylindrical shape. These tangential divisions continued throughout the culture period until prenodular and nodular structures were formed (Figs. 1–3, arrows). Starch accumulation is noted mostly in cortical parenchyma cells (Fig. 3).
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After 15–19 d in culture, the entire explant was almost covered by masses of cells that had partially individualized from the original explant. Cambial cells kept dividing tangentially into the cortical region and ultimately differentiated into vascular bundles (Figs. 8, 9, arrows). Communication between these vascular bundles and the original vascular system was not observed (Figs. 8, 10, 23, arrows). Vascular bundles organized as vascular centers were surrounded by layers of cambial cells (Fig. 8 arrows). This way, several prenodular structures were formed inside the calluses (Figs. 8, 10). At this stage, the sclerified cell layer was no longer present in explant regions where calluses showed prenodules (Fig. 8). Starch accumulation was very high in most prenodular cells and not in those cells surrounding prenodular structures (Fig. 10, arrows).The prenodular structures increased in size. The greatest extent of starch accumulation was observed within these structures (Fig. 12).
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). In fact, nodules obtained from Humulus lupulus var. Nugget internodal explants showed a central vascular area surrounded by cambial cells and a cortical parenchyma area surrounded by elongated cells (Figs. 11, 14). These elongated cells had their origin from cortical explant cells. These cells increased in volume due to an increase in their degree of vacuolation and aligned successively at the periphery of prenodular and nodular structures as these were growing (Fig. 11). Several prenodules and, thus, nodules could arise from the same explant in different directions (Figs. 7, 11). Starch mobilization started in these nodules before shoot regeneration (Fig. 13). Control explants cultured for 8, 15, or 28 d on basal medium also accumulate starch (Fig. 19) but at a lower extent than explants cultured in the presence of growth regulators (Fig. 6). Insignificant calluses appeared sporadically in these explants (Fig. 27), but they did not evolve throughout the culture period. Nodule formation and shoot bud regeneration were never obtained from internodal explants cultured on MS medium without growth regulators.
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Several nodules showed multiple vascularized centers around which could occur nodule separation into "daughter nodules" (Figs. 18). These newly formed nodules increased in volume and could regenerate shoot buds all over their surface (Figs. 24, 25, 26).
DISCUSSION
Histological studies
Our observations indicate that shoot buds in Humulus lupulus var. Nugget arose from organogenic nodules derived from explant internodes. This process has already been described for several species (Aitken-Christie, Singh, and Davies, 1988
; McCown et al., 1988
; Warrag, Lesney, and Rockwood, 1991
; Piéron, Belaizi, and Boxus, 1993
; Teng, 1997
). Nodule formation has also been obtained for Humulus lupulus var. Eroica by using petioles as explant source (Batista et al., 2000
). The differences in organogenic response of different types of explants from several hop varieties revealed a strong genotypic influence (Batista, Sousa, and Pais, 1996
). Intraspecific differences in organogenic response have been reported earlier (Narasimhulu and Chopra, 1988
; Julliard et al., 1992
). However, both histological studies and starch accumulation/mobilization cycles revealed that the regeneration process in Humulus lupulus var. Nugget from petioles is similar to that reported above for internodes as well as to regeneration processes occurring in other hop varieties (data not shown).
The organogenic process starts with some callus formation. Several prenodular structures could appear within one callus and even separate from each other before they give rise to nodules. Nevertheless, histological observations could not ascertain whether this process of separation of prenodular structures occurred at this stage. These structures were established 15–19 d after culture initiation, and between 10 and 20 d of culture some cells undergo a self-perpetuating change that commits them to organogenesis (Fortes and Pais, unpublished data). Thus, it is likely that once these prenodular structures are established, shoot-bud regeneration will occur even in the absence of growth regulators.
A transdifferentiation of isolated Zinnia elegans mesophyll cells into tracheary elements has been recently described (Fukuda and Komamine, 1980
; Stacey et al., 1995
). However, the results on hop (var. Nugget) presented here support a cambial origin for vascular centers of prenodular structures instead of an origin based on a particular transdifferentiation process of prenodular parenchymatous cells. Moreover, sclerified cortical cells were no longer visible in explant areas where calluses showing prenodular structures were formed. This is probably because cambial cells started dividing in the cortical region and callus tissue. Some of these could ultimately differentiate vascular elements. This sclerified layer has been already described during shoot organogenesis from stem explants of Brassica napus (Julliard et al., 1992
) and seemed to isolate the actively growing cortical cells from the internal pith, a role also played by this sclerified layer on cultured Humulus lupulus var. Nugget explants.
Prenodules and, thus, nodules have a cambial origin and arise without significant callus formation. Thus, it is probable that the genetic fidelity of the hop plantlets regenerated from these nodules was maintained. In fact, this feature is a major prerequisite for stable genetic transformation. Buds formed within callus or callus-derived structures can show ploidy changes (Thorpe, 1982
).
Nodules obtained from Humulus lupulus var. Nugget internodal explants showed a central vascular area surrounded by cambial cells and a cortical parenchyma area surrounded by elongated cells. The function of these elongated cells is not clear. They may keep nodules tightly attached to the original explant while contributing to their independency since they surround the entire nodular structure. However, once preliminary stages of shoot bud regeneration were initiated these cells started to disappear around nodules and were no longer present in those nodular areas where the regeneration process was occurring.
After 3–4 wk of culture, nodules had greatly increased their volume due to divisions taking place in cambial cells surrounding vascular centers. Most nodules showed multiple vascularization centers ("polycenter nodules" according to McCown et al., 1988
) around which nodulation could occur and form small "daughter nodules." This separation process seems to be initiated by the formation of a necrosis layer at the future place of nodule separation (Aitken-Christie, Singh, and Davies, 1988
; Piéron, Belaizi, and Boxus, 1993
). Each of these "daughter nodules" could grow more and then undergo organogenesis which would greatly increase the overall number of regenerated shoot buds obtained per internodal explant. This high regeneration level is also an important feature if genetic transformation is desirable (McCown et al., 1991
). In addition, histological observations revealed that both processes (nodule separation and shoot bud regeneration) started ocurring after 32–45 d in culture. This can be explained by the fact that if all of the prenodular structures originally formed ultimately gave rise to nodules, it is likely that some nodules were regenerating shoot buds after 32–45 d of culture, while others were separating into small nodules. In conclusion, both processes were probably taking place simultaneously.
Activation of epidermal and subepidermal cell divisions leading to shoot bud regeneration only occurred in nodular areas facing vascular bundles. This is not surprising since a neovascularization is established between the nodule's vascularization center and the newly formed leaf primordia. This process of neovascularization has been reported earlier by Piéron, Belaizi, and Boxus (1993)
for Cichorium intybus nodule culture.
By the time shoot bud formation was occurring from nodules or as soon as they had reached a critical volume, densely stained cambial cells and surrounding vascular centers were no longer visible. It seems that vascular bundles, forming these vascular centers started dispersing all over the nodule's structure and thus several neovascularizations were being established in several nodular areas. Epidermal and subepidermal cell divisions occurred, as stated above, in several nodular areas, giving rise to a high number of plantlets formation. This activation of cell divisons in different regions might be due to positional effects carried out by neighboring cells. In fact, physical influences generated by neighboring cells have recently been shown to be a valuable contributor to cell differentiation (Westhoff et al., 1998
).
Histological study of control explants showed that they could develop small prenodular structures despite the absence of growth regulators. However, nodules and their characteristic cell and tissue differentiation were never observed on these explants. This is not surprising since vascularization centers could not arise without auxin and cytokinin addition, substances known to interfere with vascular element formation and cell division. Finally, the fact that shoot bud regeneration was never observed in control explants confirmed nodule formation as a crucial step in the organogenic process of internodal explants of Humulus lupulus var. Nugget.
Changes in the starch content
The heavy accumulation of starch in shoot-forming tissues has been reported in several studies (Thorpe, Joy, and Leung, 1986
; Mangat, Pelekis, and Cassels, 1990
; Redway, 1991
; Julliard et al., 1992
) and seems to precede any formation of shoot primordia. Internodal explants of Humulus lupulus var. Nugget showed a large deposition of starch after being cultured for 7–9 d on medium with growth regulators. A rapid and observable increase in starch was also detected in the explant tissues cultured solely on basal medium. This should be expected since both media are sucrose enriched. However, starch accumulation in control explants was detected at a lower extent than in explants cultured under organogenesis-inducing conditions. Moreover, the amount of starch in control explants was not depleted throughout the culture period (data not shown), whereas cells of the developing shoot primordia and the subjacent nodular tissue revealed a noticeable decrease in their starch content. This cycle of starch accumulation/mobilization reflects a potential causative role of starch in organogenesis and suggests that starch is used both during organ initiation and later in its development.
Organogenesis is a high-energy-requiring process. Starch degradation results in the formation of glycolytic intermediates that will subsequently be catabolized and yield high amounts of ATP (Mangat, Pelekis and Cassels, 1990
). The oxidative catabolism of starch during the organogenic process can be corroborated by the detection of a continued increase in oxygen uptake. This has been reported by Jensen (1962)
for embryo development in cotton. In fact, embryogenesis also seems to be a morphogenic process related to a preliminary phase of heavy starch accumulation (Williams and Maheshwaran, 1986
; Stamp, 1987
; Brisibe et al., 1993
).
The importance of starch accumulation in shoot regeneration was emphasized by Thorpe and Murashige (1970)
and Thorpe, Joy, and Leung (1986)
. These researchers found that addition of gibberellic acid (GA3) to the culture medium suppressed the high accumulation of starch required for shoot formation, probably by stimulating 
-amylase synthesis and, thus, starch hydrolysis. The premise that a certain concentration of starch is necessary for organogenesis is further suggested by the results presented here. In fact, control explants accummulated less starch than explants cultured on medium with growth regulators and organogenesis was never obtained in the latter explants.
In conclusion, the present results and those of others (Thorpe and Murashige, 1970
; Ho and Vasil, 1983
; Thorpe, Joy, and Leung, 1986
; Stamp, 1987
) strongly support a causative role of starch in organogenesis.
FOOTNOTES
1 The authors thank Mr. Chaveiro for technical assistance in SEM. This work was supported by PRAXIS XXI programme (project PRAXIS/ 2/ 2.1/ BIO/ 1142/ 95 and grant BIC/ 3109/ 96). 
2 Author for correspondence (mfortes{at}fc.ul.pt
).
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