3. RESULTADOS Y DISCUSIÓN
3.6. RESULTADOS ANTE ERRORES EN EL MODELO
Germ ination using ethylene as a stim ulant was easily scaled up (section 3.2.1). Two batches of Striga seeds (125 mg) were germinated with ethylene in 10 gas tight vials. The germinated seeds were then collected on a 2.5 cm filter paper disc using a millipore filter unit and gentle suction. The seedlings were washed off the filter into 50 ml 10-5M 2,6-DMBQ in 250 ml conical flasks. Both flasks were incubated at 33 OC in the dark for 12 h, one underwent gentle shaking on the shaking platform of an incubator while the other was shaken vigorously at 180 rpm in an o rb ital shaker.
Percentage haustorial induction was determ ined by counting the num ber of seedlings which showed radicle swelling with the developm ent of hairs. Three aliquots of approximately one ml were transferred from each treatment to a slide and all seedlings in the field of view of a dissection microscope at magnification 15X were examined. The results are presented in Table 3.1.
Table 3.1
H austorial induction rates of S. hermonthica in response to 2,6-DMBQ after 12 h 33oC.
Treatment Gentle O rb ita l
shaking s h a k in g
% H austorial
I n d u c tio n 0 75
The germinated S. hermonthica seedlings which underw ent gentle agitation throughout exposure to 2,6-DMBQ showed no incidence of haustorial induction. This was attributed to the high respiration rates of the seedlings (see below, section 4.2.3) It was thought that the oxygen requirem ent of the seedlings outstripped the diffusion rate of oxygen into the 2,6-DM BQ solution, thereby inhibiting seedling growth and haustorial d evelopm ent.
The seedlings which were shaken at 180 rpm had a percentage haustorial induction rate of 75 %, vigorous shaking appeared to aerate the medium sufficiently for growth to continue.
s.
hermonthica seedlings were periodically removed to assess the timing o f haustorial developm ent (figures 3.5 and 3.6).Figure 3.5 shows the sequence of haustorial developm ent. After three hours it was obvious by eye that the Striga radicles had ceased elongation in response to 2,6-DMBQ when compared with water treated controls (fig. 3.5B). Rapid changes in the radicle apex followed, with a shift to radial expansion of the cells of the root tip (fig. 3.5C). This reorientation of cell expansion was closely followed by initiation of haustorial hairs, evident in most seedlings by 5 h. Because of the rapidity of haustorial developm ent it was difficult to tem porally separate the onset of radial enlargement from hair initiation (fig 3.5C and D). The development of haustorial hairs was acropetal, arising from files of cells in the epidermis of the radially expanding radicle tip. The haustorial hairs elongated rapidly and were obvious by 6 h after the start of 2,6-DMBQ treatment (fig. 3.5E) and by 7.5 h the majority of seedlings had a fully developed primary haustorium at the radicle tip (fig. 3.5F).
Figure 3.5
H austorial developm ent of S. hermonthica seedlings in vitro. A seedling after germination treatment (A) and after 3 h in water (B) showed increased radicle elongation. Seedlings incubated in 2,6-DMBQ ceased radicle elongation and cells at the radicle tip began to radially expand by 4 h (C) and haustorial hair initials were observed by 5 h (D). H austorial developm ent proceeded with increased haustorial hair length by 6 h (E) and full development by 7.5 h (F). Scale b a r = 0 .2 5 m m . [
The difference in length of seedling radicles which were
haustorially induced for 7.5 h compared to water treated controls was striking (figure 3.6). In future experiments, 7.5 h was used as the time required for full haustorial development of S. herm onthica to occur in
vitro. S trig a seedlings left in 2,6-DMBQ for longer periods of time did not develop any further than those incubated for 7.5 h, except for an
increase in haustorial hair length and slight lengthening of the h a u s to r i u m .
In the large scale in vitro 2,6-DMBQ treatment, haustorial
developm ent is com parable with haustorial form ation in the parasitic angiosperm s. H owever, haustorial developm ent in vitro occurred in a much shorter time period than has been recorded before. S. asiatica showed prim ary haustoria 24 h after treatment with 2,6-DM BQ at tem peratures of 28 oC (Lynn and Chang 1990; Wolfe and Timko 1991). The more rapid appearance of haustoria in S. herm onthica could reflect the different growth temperatures used. However, work by Lane et al. (1991) dem onstrated that infection of maize and sorghum roots was much slow er in S. asiatica than in S. hermonthica. This indicates that there is a real difference in the rate of haustorial ontogeny between the two species.
F ig u re 3.6
Comparison of seedlings treated with water or 2,6-DMBQ in the large-scale haustorial induction system. Appearance of a seedling
incubated in water for 7.5 h after germination treatment (A) compared to a seedling treated with 2,6-DMBQ for 5 h (B) and for 7.5 h (C) after germ ination treatm ent.S cale b a r = 0.25 mm .
The rapidity of haustorial development of S. herm onthica in the optim ised growth system implies that ceil expansion rather than an increase in cell number produces the characteristic swelling of the radicle. Sim ilarly, Riopel and Musselman (1979) noted that the young haustorium of A. purpurea results principally from the enlargem ent o f existing cells of the cortex and epidermis rather than by an increased num ber of cells.
The apparent cessation of elongation in parasitic plant radicles exposed to haustorial inducers may be attributed to the redirection of cell grow th at the radicle tip from longitudinal to radial expansion. The shape of a growing plant cell is determined by the organisation of the cellulose microfibrils of the cell wall, the driving force for expansion being turgor pressure. Cellulose microfibrils are unstretchable and m ust slide past each other to allow the cell to grow. In elongating cells the m ost recently laid down microfibrils in the side walls com m only lie perpendicular to the axis of elongation, surrounding the cell with
num erous hoops of cellulose. The oriented hoops of cellulose m icrofibrils prevent m ajor increases in the width of a growing cell, while perm itting turgor pressure to cause an increase in cell length. The pattern of cell wall deposition is controlled by the pattern of m icrotubules in the cell cortex which direct the deposition of wall material from the G olgi
expansion of cells by organising the cortical m icrotubules in a direction longitudinal to the cell axis and , as a consequence, newly synthesised cellulose m icrofibrils are laid down with the same orientation (Shibaoka,
1991). As cytokinin application has been found to induce haustorial developm ent in Striga species (Riopel and Baird, 1987) and C u s c u t a species (Huang and Li, 1991), it may be that haustorial inducers cause an increase in cytokinin biosynthesis, which serves as the endogenous signal directing haustorial form ation
All S. hermonthica seedlings that responded to 2,6-DMBQ
developed haustorial hairs. The haustorial hairs seem to be involved in attachm ent to the host root (as described in Chapter 2). The haustorial hairs of many parasitic angiosperms are coated in a papillate
non-cellulosic polysaccharide (Baird and Riopel, 1984, 1983) which
appears to adhere the haustorium to the host root. This attachm ent was found to be indiscriminate and in this study S. herm onthica was also found to adhere to plastic micro titre plates and glass m icroscope slides surfaces. Thus dem onstrating the functional sim ilarity o f haustorial hairs of S. hermonthica with those of other parasitic angiosperm s.
This experim ent provided the parameters for a highly reproducible system for generating gram quantities of haustorially induced S .
h e r m o n th ic a . That is, 125 mg (original seed weight) of Striga seedlings could be haustorially induced in 50 ml 1 x 1 0 - 5 M 2,6-DMBQ at 33°C while
3.3 SUMMARY
The results from the experiments outlined in this Chapter led to the developm ent of the following method for the generation of
g erm in ated ,h au sto rially -in d u ced populations of S. herm onthica.
Up to 500 mg of preconditioned S.hermonthica seed was roughly divided between 20X 7 ml gas tight vials containing 5 ml sterile distilled water. The vials were injected with approximately 400 |il 1 % ethylene in nitrogen and rotated end to end for 13 h at 33 oC in the dark. The germ inated seedlings were collected and transferred to two 250 ml
conical flasks containing 100 ml 10-5 M 2,6-DMBQ. The flasks were placed in a rotary shaker at 180 rpm at 33 °C in the dark for 7.5 h. The
seedlings were harvested in a millipore filter unit onto a 2.5 cm diam eter disc of IMM Whatman filter paper. The seedlings were either; quickly scraped into a precooled mortar and ground to a fine powder in liquid nitrogen and transferred to a 15 ml falcon tube and stored at -80 °C u n til required, or ground up in a protein extraction buffer at 4 °C. C ontrol
seedlings were given a water treatment for 7.5 h under otherwise identical conditions. If smaller amounts of S. herm onthica seed were used, volumes of water and 2,6-DMBQ solution were scaled down a c c o rd in g ly .
C h apter 4