1. Introduction:
2. Lotus Activities: reports and abstracts
2. Curent Listing of Lotus Newsletter Recipients.
Purpose: The Lotus
Newsletter consists of informal communications of research
information on Lotus. Reports of any phase of research
on Lotus breeding, genetics, taxonomy, management, utilization
or physiology are welcome. Your biographic sketches and information
about your research objectives, approaches, and progress including
titles of your publications are encouraged. Seed requests and
news items are accepted.
This is the 25th year of publication for the Lotus
Newsletter. Now is the time to consider contributing to
the 26th volume of the Lotus Newsletter.
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Lotus Newsletter
Dr. P. R. Beuselinck, USDA-ARS
Plant Genetics Research Unit
207 Waters Hall
University of Missouri
Columbia, MO 65211 U.S.A.
E-Mail pbeuselinck@plantsci.missouri.edu
FAX 573-882-1467
The expense of publishing the Lotus Newsletter has been partially covered by unrestricted research support. This issue of the Lotus Newsletter is provided to you without charge. I will continue to strive for financial support of the Lotus Newsletter to provide you with an unencumbered communication resource.
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The illustration on the cover is of a Lotus spp. L. graciously provided by Ana Arambarri (Argentina) . The illustration of L. unifoliatus Benth. (syn. L. purshianus) is the third in a series of illustrations that started with L. edulis in Volume 23.
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Second Conference on Forage Quality, Evaluation, and Use. 13-15 April 1994. Lincoln, Nebraska. Contact Dr. Lowell Moser (402) 472-1558 for further details.
XVIII International Grasslands Congress. June 8-19 1997. The Congress will be split between two locations: i) Winnipeg, Manitoba and ii) Saskatoon, Saskatchewan Canada. For more information contact: Box 4520, Station C, Calgary, Alberta T2T 5N3. Voice: (403) 244-4487; FAX (403) 244-2340.
G.M.Tourn, A. Bartoli, R.D.Tortosa, G. Roitman and M.C.Saucede.
SUMMARY
A morphological and architectural study was made on 2 herbaceous
species of Fabaceae, Medicago sativa L. {lucerne grass)
and Lotus tenuis Wald. et Kit {narrow birdsfoot trefoil)
during the first year of growth (establishment ). These species
are perennial widespread pasture legumes We compared and discussed
the architectural unit (morphological and functional determination
of whole shoots from germination to establishment )and the variability
on different swing dates. Both species have a primary with orthotropic
basitoncal vegetative branches carried foliage leaves with spiral
arrangement. Adventitious vegetative shoots arising from the hypocotilar
region and on the nodal base second order order branches were
observed. The reproductive branches are lateral and acrotonic.
The architectural unit had no modification with different sowing
dates, but the growth dynamic was visible affected.
Additional Keys Words: architectural analysis, architecture, establishment,
Fabaceae, forage legumes, Lotus tenuis, lucerne grass,
Medicago sativa, narrow birds-foot trefoil.
G.M.Tourn and G.G.Roitman
SUMMARY
A morphological and architectural study of regenerative strategies
was made on a herbaceous species of Fabaceae, Lotus tenuis
Wald. et Kit (narrow birds-foot trefoil) growing in a widespread
pasture legume with sympodial and modular growth. We compared
and discussed the architectural model, reiterations and 2 different
regenerative strategies (rhizomes and seeds). The orthotropic
branches, by internode increases, during the growing season, changes
the orientation; it was plagiotropic at the base to orthotropic.
Some of this branches, produces adventitious roots, at nodes,
become in an adaptive reiteration. After seed dispersion, cessation
of the meristematic activity occurs. The orthotropic vegetative
aerial shoots (reiterations) grow out from the axillary buds of
the rhizomes, in the next growing season.
Keys words: architecture, branching, Fabaceae., forage legumes, Lotus tenuis, narrow birdsfoot trefoil, regenerative strategies, reiterations , rhizome.
Niizeki M. and T. Kodaira
Laboratory of Plant Breeding, Faculty of Agriculture
Hirosaki University, Hirosaki, Aomori-ken 036, Japan
It is assumed that somaclonal variation occurs, due to the lack
of some genetically controlled mechanisms in cultured calli or
cells. It is probable that these somaclonal mutations accumulate
from one cell cycle to the next. Birdsfoot trefoil (Lotus corniculatus
L.), cv. Viking is a suitable plant for the investigation
of somaclonal variation because of the high totipotency found
in its cultured cells. This is true, even when they are derived
from a single protoplast. Therefore, it is a useful plant for
comparative studies between protoplast- and seed-derived populations.
In our study (Niizeki et al. 1990) it was shown that somaclonal
variation is very useful in the improvement of quantitative traits
in this species. The populations of P2 and P3
generations were obtained by the open pollination of a regenerated
P1 protoclonal population and of a P2 population,
respectively. Seven traits indicated in Table 1 were investigated
in the P1, P2 and P3 generations.
Few significant differences were found among the three generations
in each trait, with the exception of dry matter yield and pollen
fertility. There was a drastic increase in dry matter yield in
P2 which then decreased in P3. The low yield
for P1 may have been caused by the low number of shoots
that grew on the poorly developed crown root of the regenerated
protoclones. That of P3 may have been caused by the
low number of shoots and the short plant height caused by underaverage
temperatures in the summer season of 1993. While the large number
of shoots made it substantially impossible to count them each
year, plant height was investigated in P2 growing in
both 1992 and 1993 (Table 2).They were significantly shorter in
1993 than that of 1992. In regard to pollen fertility, Niizaki
(1993) showed that it drastically increased in P2 and
P3. This may have been caused by the elimination of
gametes with abnormal chromosomal configurations in the P1
protoclones.
With regard to major genes, the nuclear DNAs were analyzed by using 2 restriction enzymes, HindIII and BamHI, and the major genes, a small subunit of RuBisCO, phenylalanine ammonialyase and ribosomal DNA, pRR217. In this experiment, it was found that these genes were very stable, without any variations. However, heterochromatin parts consisting of satellite DNA revealed a considerable number of variations in a Southern blot analysis using restriction enzymes, HindIII and EcoRI, and a probe of (GGAA)3. From these results, the two alternative assumptions considered are as follows:
Table 1. Mean values on seven traits of three generations after
regeneration from single protoplast-derived callus
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | |
| cm | cm | cm | cm | cm | cm | cm | |
| P1 | 28.2a | 2.5a | 1.4a | 11.0a | 5.8a | 8.3a | 58.5a |
| P2 | 28.9a | 2.9a | 1.5a | 12.0a | 7.5a | 17.3b | 77.1b |
| P3 | 27.3a | 2.8a | 1.5a | 11.4a | 7.3a | 9.6a | 75.0b |
1: Plant height , 2: Length of internode, 3: Stem diameter, 4: Leaflet length, 5: Leaflet width, 6: Dry matter yield, 7: Pollen fertility. Two values with different letters in the same column differ at 5% level after Duncan's multiple range test.
Table 2. Values on six traits of P2 generation in 1992 and 1993
| 1 | 2 | 3 | 4 | 5 | 6 | |
| cm | cm | mm | mm | mm | g | |
| 1992 | 28.9+5,4 | 2.9+0.5 | 1.5+0.2 | 12.0+1.3 | 7.5+1.0 | 17.3+5.4 |
| 1993 | 25.1+4.3 | 2.9+0.7 | 1.4+0.3 | 12.3+1.8 | 7.3+1.4 | 7.4+1.8 |
| t-value | 3.03 | 0.31 | 0.90 | 0.90 | 0.76 | 9.42 |
| 0.001<P<0.01 | n.s | n.s | n.s | n.s | P<0.001 |
1-6 : The same traits as those in Table 1.
REFERENCES
M. Niizeki, R. Ishikawa and K. Saito 1990. Variation in a single protoplast and seedderived population of Lotus corniculatus L. Theor.Appl. Genet.80 : 732736.
M. Niizeki 1993. Chromosomal mutation induced by protoplast culture
in Lotus corniculatus L. Lotus Newsletter 24:44.
O. S. Correa and A.J. Barneix.
Cátedra de Microbiología. Facultad de Agronomía
Buenos Aires. República Argentina
Soil acidity determines a low efficiency of the Rhizobiumlegume
symbiosis and the need to select acidtolerant symbiotic
associations. The degree of pH tolerance depends upon the bacterial
strains and the plant species involved. The bacteria can be more
sensitive to low pH than their legume host, so that their ability
to colonize acid soils is limited by the effects of acidity on
their survival and growth (O'Hare et al, 1994).
The symbiotic association between Lotus tenuis and Rhizobium
loti is successfully used on heavy and alkaline soils and
could be used also under acid conditions. Rhizobium strains
which nodulate Lotus sp. show marked differences in their
response to acidity (Wood et al, 1988) but little is known about
the effect of soil acidity on Lotus tenuis.
Our aim is to determine the response of Lotus tenuis cv Chaja
and several Rhizobium loti strains to acid pH in order
to use them under this conditions.
BACTERIAL STRAINS AND GROWTH MEDIA
Rhizobium loti strains LL22, LL32, LL54, LL55 were provided
by the Instituto Nacional de Tecnologia AgropecuariaINTACastelar,
Buenos Aires, Argentina, and Al was isolated by our laboratory.
Cultures were maintained at 4°C on yeast extract mannitol
agar (YEMA,Vincent 1970) slopes and grown in yeast extract mannitol
broth (YEMB, Vincent 1970) before using. Cultures were inoculated
(ca 105 viable cells/ml) into flasks of YEMB adjusted
to pH 4.0, 5.0, 6.0 or 7.0 with 0.1 N CIH or NaOH before autoclaving.
Flasks were held at 30°C for 48 hours on a rotary shaker
(150 rpm) and samples were withdrawn aseptically for the determination
of absorbance at 600 nm. Each experiment was performed in triplicate.
GROWTH OF LOTUS TENUIS IN ROOTING SOLUTION WITH N
Seeds of Lotus tenuis cv Chaja were surface sterilized,
washed and germinated on water agar for 2 days at 22°C before
being aseptically transferred to tubes (190 x 19 mm) containing
25 ml of sterile rooting solutions, adjusted to pH 4.0, 4.5, 5.0,
6.0 or 7.0. The rooting solution has the following composition:
Solution A: KNO3 0.1 g; MgSO4. 7H2O
0.2g; CaCl2 0.25g; FeEDTA, 10 ml of a solution containing
0.8g disodium ethylenediamine tetra acetate, 3.0 ml of a 10% solution
of FeCl3 and 11 of distilled water; trace elements
(B 0.5mg; Zn 0.05mg; Mo 0.05mg; Cu 0.02mg); deionised water 900ml.
The solution was adjusted to the required pH value with diluted
HCI or NaOH. Solution B: For pH 4.0, 4.5, 5.0 or 6.0 media 100ml
of a mixture of citric acid 0.1 M and Na2HPO4
0.2M; for pH 7.0 medium 100ml of TrisHC1 0.1M and Na2HPO4
0.2M. The two solutions were sterilized separately by autoclaving
at 121°C for 15 min. and then combined aseptically to set
the required pH.
Seedlings (1 per tube) were supported by a roll of filter paper
and the tubes were closed with cotton wool plugs. The tubes were
maintained in a controlled environment chamber with 25°C,
16 h day and 8 h night. After 50 days the plants were removed
from the rooting solution, dried for 48 h at 80°C and weighed.
Four replicates were included per treatment.
NODULATION AND GROWTH OF LOTUS TENUIS IN N-FREE ROOTING SOLUTION
Nodulation was assayed in the same conditions as described above
except no nitrogen was added to the rooting solution (KCI 0.1
g instead of KNO3) and the seedlings were inoculated
with ca 107 cfu of a single strain that were grown
in YEMB. After 50 days the plants were removed from the rooting
solution, the nodules were counted and then the plants were dried
and weighted. There were four replicates per strain and pH.
Analysis of variance was performed on data.
RESULTS AND DISCUSSION
All 5 strains tested grew at pH 5 or above (Fig. 1)
No strain grew at pH below 5.
The growth of Lotus tenuis in rooting solution with nitrogen at the different pH values showed no significant differences. This result indicates that Lotus tenuis cv Chaja is very acid tolerant when it grows in rooting solution with mineral nitrogen.
However, when inoculated, Lotus tenuis cv.Chaja in nitrogenfree
rooting solution significant differences (p<0.05) were observed
among the strains at the different pH tested. The plants inoculated
with LL22 showed the highest growth at pH 4.0 and 7.0 (Fig. 2).
The strain LL22 formed a significantly higher number of nodules
(p<0.05) than the other R. loti strains at the lowest
pH value. The nitrogen fixation by Lotus tenuis was strongly
affected by the medium pH, and the pH tolerance was dependent
upon the bacterial strains.
These results indicate that for the R. loti strains tested
in these experiments there is no relation between the ability
to grow in acid medium and to nodulate in a rooting solution at
the same pH value, and the growth in liquid culture is not an
indicator of nodulating ability under acid soil conditions.
REFERENCES
O'Hara G W and Glenn A R (1994). Arch. Microbiol. 161 286292
Vincent J M (1970). A Manual for the Practical Study of RootNodule
Bacteria. Blackwell Scientific Publications. Oxford.
Wood M, Cooper J E and Bjourson A J. (1988). Plant and Soil 107:
227231.
Walter M. Kelman
CSIRO, Division of Plant Industry
CPO, BOX 1660, Canberra, Australia
INTRODUCTION
A plant breeding program based in Canberra is aimed at producing
cultivars of Lotus pedunculatus (Lotus uliginosus)
for meat and dairy production in south eastern Australia. Selection
for low condensed tannins (CT) has been practiced to maintain
bloat protection while improving the protein utilization from
the forage.
Little is known of the genetic control of CT in this species.
A cross was made between parents with contrasting levels of CT
and F1, F2, and reciprocal backcross populations were developed.
The six generations of this cross were used to provide estimates
of the pooled additive and dominance gene effects for CT.
MATERIALS AND METHODS
Parental populations: G4703 is a diploid population developed
is New Zealand with relatively low CT content. CPI 67677 is a
diploid from the Algarve region of southern Portugal and has high
CT.
Experiment Layout: Plant were grown in the field in a randomized
block design with 4 replications. In each replication the 6 generations
were present as single-row plots of 4 plants, 0.5m apart and 1.0m
between rows. The experimental unit was a single plant.
Condensed tannins: Two basal shoots were sampled from each plant
at late vegetative/early flowering stage. The shoots were oven
dried at 70°C and grounded through a 0.5 mm sieve. CT were
extracted in 70 % acetone, hydrolyzed in butanol/hydrocloric acid
(95:5 v/v)for 1 hr at 95°C and absorbance measured at 550nm.
Concentrations were expressed as %dry weight.
The genetic effects were estimated by a weighted least square
regression analysis. The suitability of the genetic model was
examined by a chi square test comparing the observed and expected
means for each generation.
RESULTS AND DISCUSSION
The additive/dominance in strong agreement with the generation
means derived from this cross and additive gene effects for CT
were significant (Table 1), indicating that
selection for lower CT should be successful. Dominance gene effects
for CT were also significant in this cross (Table 1)
and the high mean CT content of the F1 and F2 and BC1 populations
suggest the presence of nonadditive effects for high CT
production (Table 2). This information supports
the decision to continue selection for lower CT in additional
cycles of recurrent selection before the progeny testing phase
of the breeding program.
Table 1 Generation mean analysis of condensed tannins in the cross
CPI67677 x G4703.
| Condensed tannins | P(t) | |
| m | 8.244+0.147 | |
| a | 1.087+0.221 | 0.02-0.01 |
| d | 1.625+0.536 | 0.10-0.05 |
| x2(3) | 3.09 | |
| P | 0.30-0.50 |
Table 2 Generation means for condensed tannins in the cross
CPI67677 (P1) x G4703(P2)
| Generation | Observed mean | Expected mean |
| P1 | 8.3183 | 8.5185 |
| P2 | 6.2308 | 6.3433 |
| F1 | 8.1313 | 9.0563 |
| F2 | 8.2591 | 8.2436 |
| BCP1 | 9.0016 | 8.7874 |
| BCP2 | 7.8409 | 7.6998 |
R D Sheldrick, T M Martyn and R H Lavender
BBSRC Institute of Grassland and Environmental Research
North Wyke Research Station, Okehampton, Devon EX20 2SB, UK.
INTRODUCTION
The reasons for assembling a collection of Lotus species
and cultivars and screening them at North Wyke on an acid (pH
5.4), low phosphorus status (P< 10 mg -1) site have
been described in previous issues of the Lotus Newsletter
(Sheldrick and Martyn, 1991, 1992). In brief, to maintain
agricultural production from marginal grassland areas, it will
be necessary to develop progressively lower input, yet sustainable
grazing systems. On better soil types, white clover based swards
can be used, though not without regular inputs of phosphorus fertilizer
and lime (DANI, 1992). On more acid, low phosphatestatus
soils, Lotus may prove to be a better suited option (Bullard,
1992). Yet agronomic information on such basic matters as appropriate
seedrates and choice of companion grass is lacking for UK
conditions. Recently bred varieties of perennial ryegrass are
too densely tillering to form stable associations with the more
slowly growing Lotus, so an experiment was sown in 1991
to assess four possible alternative companion grasses, for assessment
under cutting. Previous work with lower trefoil but higher grass
seedrates had suggested little effect (Davies, 1969).
MATERIALS AND METHODS
As mentioned in previous articles in this newsletter, North Wyke Research Station is situated in SouthWest England (50° 45' N Lat., 3° 55'W Long.) and has a temperate maritime climate. The altitude is approximately 184m above sea level, with an average annual rainfall of 1035 mm, of which 31% falls MaySeptember. Mean air temperature in January is 4.5°C and in July 15.3°C. The soil type at the experimental site was a poorlydrained, seasonally waterlogged silty clay loam (pelostagnogley). The site had previously been under a grass fey, and soil sampling revealed pH 6.6, phosphorus 13 mg-1and potassium 70 mg -1, no further lime or fertilizer inputs were made.
The experiment was sown in July 1991. The layout comprised three
randomised blocks of plots 1.5 m X 5.0 m, with all factorial combinations
of two Lotus species (L corniculatus cv. Leo and
L. uliginosus cv Maku), four companion grasses (Phleum
pratense cv. S.48, Agrostis capillaris cv. Muster,
Festuca pratensis cv. Senu and Poa pratensis cv. Asset)
and two grass seed rates. The Lotus was sown at 10 kg ha
-1 and the grasses at 2 or 4 kg ha -1, except
for F. pratensis which was sown at 3 or 6 kg ha -1.
The inoculated Lotus seeds were thoroughly mixed with the
appropriate quantity of grass seed, and broadcast on to a harrowed
seedbed and rolled in.
No cuts were taken in 1991, though some hand weeding was carried
out. In 1992 and 1993, three cuts were made each year using a
Haldrup plot harvester (dates of cut in Table 2), set to leave
approximately 10 cm residual stubble. Samples of herbage were
dried at 85°C to determine dry matter (DM) concentration,
and others cold stored for subsequent sorting to determine proportions
of Lotus and grass DM. Lotus material was analysed
for digestibility (predicted from pepsin/cellulase solubility)
and nitrogen content (acid digestion followed by colorimetric
assay).
RESULTS
DM yields for 1992 and 1993 are shown in Table 1,
as the total of the sown species, omitting the weed fraction.
There was no effect of grass seedrate, so this variable
has been omitted from the table. In the first year, cv. Leo significantly
outyielded (P< 0.001) cv. Maku in terms of the Lotus component,
though the situation was reversed in the following year, when
the yield of cv. Leo dropped to only one third of its 1992 value,
but the yield of cv. Maku fell much less. Thus in 1993, though
DM yields of both Lotus species had fallen, cv. Maku significantly
(P<0.001) outyielded cv. Leo. The yield of the grass component
showed the reverse trend, increasing from 1992 to 1993, and compensating
for the fall in Lotus DM yield. Thus while the annual yield
of herbage from the sown species was significantly greater (P<0.001)
for cv. Leo swards than cv. Maku swards in 1992, the 1993 yields
showed no difference.
Table 1. Annual dry matter yields of sown species for 1992 and 1993 (Sown July 1991)
| Comparisons: | ||||||
| L. corniculatus cv. Leo | ||||||
| L. uliginosus cv. Maku | ||||||
| s.e.d. (30 residual df) | ||||||
| Level of significance | ||||||
| Phleum pratense cv. S.48 | ||||||
| Agrostis capillaris cv.Muster | ||||||
| Festuca pratensis cv. Asset | ||||||
| Poa pratensis cv. Asset | ||||||
| s.e.d. (30 residual df) | ||||||
| Level of significance | ||||||
There were no significant interactions between the Lotus spp
and companion grass variables in 1992, though there was in 1993
(P<0.05), due to poor performance of the cv. Leo and P.
pratensis cv. Asset combination in comparison with all others.
Within the companion grass comparison, the highest Lotus component
yield came from mixtures with cv. Asset in both years. However,
cv. Asset gave the lowest sown grass DM yield and total annual
yield of sown species in both years. Lotus mixtures with
cv. Senu showed lower yields of sown grass than either cv. S.48
or cv. Muster in 1992 (P<0.001) but improved markedly in 1993,
so that coupled with fair yields of the Lotus component,
total yields of the sown species were not significantly different
from either cv. S.48 or cv. Muster.
The forage quality data shown in Table 2, confirmed
previous results (Sheldrick and Martyn, 1992) that L. corniculatus
had a higher digestibility then L. uliginosus, but
a lower nitrogen content.
Table 2 Lotus forage quality data at three cuts, 1992 and 1993
| DOMD | Tot N | DOMD | Tot N | DOMD | Tot N | ||
| 1992 | |||||||
| L. corniculatus cv. Leo | |||||||
| L. uliginosus cv. Maku | |||||||
| s.e.d. (30 residual df) | |||||||
| Level of significance | |||||||
| 1993 | |||||||
| L. corniculatus cv. Leo | |||||||
| L. uliginosus cv. Maku | |||||||
| s.e.d. (30 residual df) | |||||||
| Level of significance | |||||||
| DOMD = digestible organic matter as a percentage of total DM | |||||||
| Tot N = total nitrogen content as g/kg of DM | |||||||
In both years there was a trend towards higher forage quality
at the late autumn cut, in respect of both digestibility and nitrogen
content. However, this cut was also the lightest in term of Lotus
yield, so the net effect on annual herbage quality will be
small. The lower digestibility of L uliginosis is to be
expected in any enzymebased assessments, of course, because
of the much higher levels of condensed tannins that this species
contains (Roberts and Beuselinck, 1992).
DISCUSSION AND CONCLUSIONS
As found previously in our screening trial (Sheldrick and Martyn,
1991, 1992), yields of L uliginosus were lower than those
of L corniculatus initially, possibly due to resources
being diverted to stolon development in the former species. The
sharp decline in yield of cv. Leo in 1993 did not appear to be
due to pest or disease attack, and might have resulted from competition
by the increased grass growth supported by the nitrogen fixed
the previous year.
Although the Lotus x companion grass interaction did not
give any clear cut indication of superior combinations, it would
appear that F. pratensis cv. Senu has generally combined
well with both Lotus species, allowing good growth of the
legume component and hence likely to provide sustained high yields
of the mixture.
The experiment has continued during 1994, and some answers to
the questions raised may become apparent when the three years
results are assessed.
FUTURE RESEARCH
It has not yet proven possible to start the grazing experiment
that was mentioned in the 1992 Newsletter (Sheldrick and Martyn,
1992). However, some limited progress may be possible in 1995,
as experience of grazing management for Lotus swards is
totally lacking in the UK. Indeed, it is not known whether grassLotus
associations can survive under meaningful animal stocking
levels. Properly researched guidelines for the management
of Lotus could enable this legume to provide a valuable
alternative to white clover based technology for marginal land
situations.
REFERENCES
Bullard, M.J. (1992) The potential of birdsfoot trefoil (Lotus
corniculatus L.) for U.K. agriculture. D.Phil. Thesis,
University of York.
DANI (1992) Clover: a guide for use on the farm. Department
of Agriculture for Northern Ireland. HMSO, 36pp.
Davies, W.E. (1969) The potential of Lotus spp. for hill
land in Wales. Journal of the British Grassland Society, 24,
264 270.
Roberts, C.A. and Beuselinck, P.R. (1992) Condensed tannins in
Lotus Species. Lotus Newsletter, 23, 41.
Sheldrick, R.D. and Martyn, T.M. (1991) Progress with screening
Lotus species and varieties on an acid, lowphosphate soil
type in UK. Lotus Newsletter, 22, 3739.
Sheldrick, R.D. and Martyn, T.M. (1992) Further development with Lotus screening in the UK. Lotus Newsletter, 23, 37-40.
Denise E. Cooke and K. Judith Webb
Institute of Grassland and Environmental Research
Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB, UK
INTRODUCTION
The gus gene
has been introduced successfully into tobacco, and the extraction,
assay and staining of the GUS enzyme has been optimised for this
plant (Jefferson et al.,
1987). We aim to investigate if the findings of
Jefferson and coworkers are similar for Lotus
corniculatus.
Unlike tobacco, transformed L. corniculatus
roots accumulate phenolic compounds when grown
in culture (Morris & Robbins, 1992). These phenolic compounds
are released when the tissue is homogenised, and are capable of
forming chemical bonds with proteins (Loomis 1974). Hence, these
compounds may interfere with the extraction of GUS from L.
corniculatus root
cultures. Polyvinylpyrrolidone(PVP), or the anionexchanger
Dowex can be used to remove phenolic compounds (Loomis 1974; Robbins
et al. 1991).
Therefore, we aim to assess if these compounds should be included
in the extraction procedure.
Jefferson et al. (1987)
found that there was no intrinsic activity in tobacco which could
interfere with the GUS assay. However, a number of plant species
contain an intrinsic GUSlike activity (Hu et
al. 1990) with an optimum of pH 5 (Alwen et
al., 1990), which may interfere with the assay
of the E. coli GUS.
Therefore, we aim to assess if L. corniculatus
contains a similar activity.
MATERIALS AND METHODS
Two lines of L. corniculatus
(bird's foot trefoil) cv Leo (6 and 12) which
expressed the gus gene, and a control L. corniculatus
Alc20.2 which did not contain the gus gene,
were available (Webb et
al. 1994).
Roots were frozen in liquid nitrogen and ground to a fine powder
using a mortar and pestle. Precooled extraction buffer was added
and the mixture was centrifuged to remove the debris. The fluorimetric
GUS assay, and histochemical staining with Xgluc, were performed
according to Jefferson et
al. (1987). E. coli
GUS (Boehringer Mannheim) was used as a positive
control and extraction buffer as a negative control. The strongly
basic anion exchanger Dowex 1 (chloride, Sigma), and polyvinylpyrrolidone
(PVP360, Sigma), were included in the extraction buffer
at 5%(w/v), and SmM saccharolactone (Sigma) was included
in the assay, where indicated.
Analysis of variance (ANOVA) was carried out with the Genstat
program (Payne et al. 1987).
Scheffe's multiple comparison procedure (the S test) was performed
according to Scheffe (1953).
RESULTS AND DISCUSSION
EXPRESSING GUS ACTIVITY: Jefferson et
al. ( 1987) expressed GUS activity on a protein
or DNA basis. However, we found it impossible to express GUS activity
on a DNA basis because it is very difficult to extract DNA from
L. corniculatus
(Robbins et
al. 1991). When expressing GUS activity on a protein
basis, it is important to determine the protein concentration
within hours of the GUS assay because the protein concentration
in samples stored at 4°C were reduced by 719% overnight
and by 1749% after 3 days. Alternatively, GUS activity can be
expressed on a fresh weight basis.
EFFICIENCY OF EXTRACTION OF GUS FROM ROOT CULTURES: To
investigate if GUS in the root tissue was being completely recovered
in the soluble extract, the GUS activity was measured in the extract,
in four subsequent washes of the debris and in the remaining debris.
The GUS activity in each
Table 1: Extraction of GUS from L. corniculatus roots.
fraction, as a percentage of the total measured activity, is shown
in Table 1. Approximately 98% of the measured GUS activity was
present in the soluble extract, suggesting that the GUS enzyme
is effficiently extracted from root tissue.
THE USE OF DOWEX AND PVP TO PREVENT THE FORMATION OF PHENOLICPROTEIN
COMPLEXES: When Dowex was used in the extraction of GUS from root
cultures, no GUS activity was detected in the supernatant using
the fluorimetric GUS assay. However, the debris stained blue with
Xgluc, indicating that the GUS enzyme had bound to the Dowex.
Furthermore, the addition of Dowex to a commercially available
purified enzyme solution substantially reduced the enzyme activity.
Consequently, the use of Dowex in the extraction protocol is not
recommended.
When PVP was included in the extraction protocol, there was no
significant difference in the GUS activity compared to that measured
in extracts prepared without PVP. This suggests that the phenolics
present in the root tissue did not interfere with the isolation
of GUS. As a result, PVP was not routinely included in the extraction
buffer.
STORAGE OF EXTRACTS: Jefferson et al. (1987) routinely
stored their tobacco leaf extracts at 70°C. However,
when Lotus root extracts were stored at 70°C
there was a 2040% reduction in GUS activity, possibly due
to intrinsic proteases. As a result, root extracts were assayed
for GUS as soon as they were prepared. We suggest that tissue
should be stored at 70°C until it is convenient to
measure the GUS activity.
ENDOGENOUS GUS ACTIVITY: No endogenous GUS activity was found
in L corniculatus root cultures, which did not contain
the gus gene, when analysed at pH 7, or pH 5, or when stained
with Xgluc (see Figure 1).
GUSDEPENDENT FLUORESCENCE: The fluorescence measured in
the GUS assay was shown to be due to the presence of a glucuronidase
activity by including saccharolactone in the assay buffer.
This specific glucuronidase inhibitor reduced the GUS activity
to a very low level. The inhibitor did not completely remove the
GUS activity, probably because of the inhibitor's instability
at pH7.
RECOVERY OF A PURIFIED GUS ENZYME FROM ROOT EXTRACTS: The recovery
of a commercially available purified E. coli GUS
enzyme, included in the extraction buffer when preparing extracts
from root cultures which did not contain the gus gene,
was found to be on average 82% (standard error=5). It has already
been shown that nearly all (98%) of the GUS activity is present
in the extract. Therefore the loss of enzyme must be due to inactivation;
the precise reason for which is not known. One possibility is
that proteases inactivate the enzyme. Including the protease inhibitor
PMSF (25µg/ml) in the extraction buffer did not significantly
affect the GUS activity. Since PMSF can only inhibit serine proteases,
perhaps other proteases are inactivating the GUS enzyme. However,
a percentage recovery of GUS of 82% was satisfactory.
HISTOCHEMICAL STAINING WITH XGLUC: L corniculatus root
cultures typically stained with Xgluc as shown in Figure 1.
The roots showed intense blue colour at the root tips, which faded
along the length of the root, and was often absent in the old
tissue. This staining pattern suggests that there is a high amount
of GUS activity in the root tips, which decreases along the length
of the root towards the old tissue.
(Caption for Fig. 1. - L corniculatus line 6 roots, 3,
6 and 10 days old (from left to right, at the top of the scanned
photograph) stain blue with Xgluc because they are expressing
the gus gene. L corniculatus Alc20.2 roots, 3, 6
and 10 days old (from left to right, at the bottom of the scanned
photograph) do not stain because they do not have the inserted
gus gene.)
To investigate if the staining pattern realistically reflected
the GUS activity, line 6 roots of various ages (7, 14, 21, 30
and 40 days old) were sectioned and the GUS activity was determined
using the fluorimetric GUS assay (see Figure 2,
(A) A schematic diagram of a 40 day old root. (B and C) The GUS
activity in various root sections. Each bar represents the mean
of three roots, which were grown in separate flasks, and the associated
error bars represent the standard error.). There was no significant
difference between the GUS activity measured in the different
regions of the roots and shoots, except for root tissue analysed
on day 7. In this case the GUS activity in the old tissue was
significantly higher than that in the tips.
These findings contradict the histochemical staining results,
possibly because the staining pattern reflects the diffusion into
the root of the substrate, or oxygen (needed for the dimerisation
of the product to form a blue dye). Supporting evidence comes
from the observations that 3 and 6 day old roots stain completely
blue if left for approximately two days, but 14 day old roots
do not stain very well. Therefore, care must be taken when interpreting
the results of histochemical staining particularly because a lack
of staining does not necessarily mean that there is no GUS activity
present. Similarly, Harris et al. (1990) found that although
transgenic maize callus stained nonuniformly, sectors which
did not stain blue contained levels of GUS activity which were
similar to those of blue sectors (determined by the fluorimetric
assay).
CONCLUSIONS
We found that gus can be used successfully as a marker
gene with L corniculatus in a similar manner as described
by Jefferson et al. (1987). Extraction of GUS is efficient
and does not appear to be affected by the presence of phenolic
compounds, and the fluorescence measured is due to the expression
of the E. coli gus gene since no endogenous GUSlike
activity was found in this plant. However, it is recommended that
the assays for GUS activity and protein concentration are performed
as soon as possible after the extracts have been prepared. In
addition, care must be taken when interpreting the results of
Xgluc staining.
ACKNOWLEDGEMENTS
The Institute of Grassland and Environmental Research is grantaided
by the BBSRC. This work was supported by a studentship from the
University of Wales, Aberystwyth. We would like to thank F. Potter
and S. Mizen for their assistance.
REFERENCES
Alwen A, Vicente O, HeberleBors E (1990) Use of E. cold GUS as a reporter gene in plants: possible interference of endogenous ßglucuronidases, in Abstracts Vllth International Congress on Plant Tissue and CeR Culture (Nijkamp HJJ, Van der Plas, LHW, Van Aartrijk J, eds.), p46, Kluwer Academic Publishers, The Netherlands.
Gamborg O L, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158.
Harris RR, DeRobertis GA, Pierce DA, Moynihan MR, Everett NP (1990) Heterogeneity of Xgluc staining in transgenic maize callus, in Abstracts Vllth International Congress on Plant Tissue and Cell Culture (Nijkamp HJJ, Van der Plas LHW, Van Aartrijk J, eds.), p 176, Kluwer Academic Publishers, The Netherlands.
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUSfusions: ,ßglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 39013907.
Loomis WD (1974) Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Methods in Enzymology31: 528544.
Morris P, RobbinsMP (1992) Condensed tannin formation by Agrobacterium rhizogenes transformed root end shoot organ cultures of Lotus corniculatus. J. Exp. Bot. 43: 221232.
Payne RW, Lane PW, Ainsley AK, Bicknell KE, Digby PGN, Harding SA, Leech PK, Simpson HR, Todd AD, Verrier PJ, White RP (1987) Genstat 5 Reference Manual. Oxford University Press, Oxford.
Robbins MP, Evans TE, Morris P, Carron TR (1991) Some notes on the extraction of genomic DNA from transgenic Lotus corniculatus. Lotus Newsletter 22: 1821.
Scheffe H (1953) A method for judging all contrasts in the analysis of variance. Biometrika 40: 87104.
Webb KJ, Robbins MP, Mizen S (1994) Expression of GUS in primary transformants and segregation patterns of GUS, T L and TR-DNA in the T1 generation of hairy root transformants of Lotus corniculatus. Transgenic Research 3: 232240.
B. Jorgensen, L. Skøt, and K.J. Webb
Institute of Grassland and Environmental Research
Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB
Wales, UK
INTRODUCTION
We are evaluating the legume Lotus japonicus as a candidate
for a TDNA insertional mutagenesis programme. The aim of
the programme is to identify genes involved in nodulation and
nitrogen fixation pathways.
During transformation, the TDNA of Agrobacterium tumefaciens
integrates into the plant genome, often into actively transcribed
genes. The insertion of TDNA with a promoterless GUS construct
into an active gene can then be detected by GUS activity which
can easily be seen with a histochemical reaction giving a blue
colour. The insertion of the TDNA could also give rise to
mutants.
One of the few easily transformed and regenerated legume species
is L. japonicus (Handberg & Stougaard, 1992). L.
japonicus is a small selffertile legume in the family
group of Lotus corniculatus. L. japonicus is diploid with
12 chromosomes and a genome size of 1.1 pg. The species is now
being used in many laboratories in nitrogen fixation related work.
The transformation and regeneration procedure for L. japonicus
results in a high transformation frequency and a high shoot
frequency, both of which will be necessary for a TDNA tagging
programme. One of the few disadvantages of this procedure are
the regular weekly transfers of the explants to fresh medium for
a minimum of 4 months.
This experiment evaluates the effects of alterations in the transfer
interval on the percentage of transformation. Transferring the
explants every two weeks or three weeks instead of every one week,
would make the regeneration and transformation procedure less
intensive. In addition we have studied the effect of extending
the callus induction phase from the normal four weeks to six and
ten weeks on the transformation frequency and, in nontransformed
callus cultures, on shoot regeneration.
EXPERIMENTAL
L. japonicus was transformed with LBA 4404 (pAGUSBin19)
(Topping et al, 1991 ) according to the method published
by Handberg and Stougaard (1992). The nontransformed explants
are placed on callus induction medium containing 2,4D and kinetin
for 4 weeks before transfer to shoot induction medium.
Following cocultivation, the transformed explants are transferred
to callus induction medium for one week, then transferred to callus
induction medium with selection until the callus is 12 mm
in size. The explants are then transferred to shoot induction
medium.
Here, all explants (both transformed and nontransformed)
were transferred to shoot induction media after either four, six
or ten weeks of callus induction. Both the nontransformed
and the transformed explants normally require weekly transfer
to fresh medium. In this experiment the transformed explants were
transferred to fresh medium every one, two or three weeks from
the onset of selection. Between 80100 explants were exposed
to each treatment.
The data are presented as the percentage of explants transformed
(measured as surviving callus after selection). The shoot regeneration
data in the nontransformed explants are shown as the number
of weeks before the appearance of the first callus with shoots
and the percentage of callus with shoots.
RESULTS
THE EFFECT OF INCREASED TIME BETWEEN TRANSFERS ON
THE PERCENTAGE OF EXPLANTS TRANSFORMED: The time interval
between the transfers to fresh medium was an important factor
for the percentage of explants transformed, as can be seen in
figure 1. The percentage of transformation declined when transfer
intervals were longer than one week. The percentage of explants
transformed are 6070% for transfers every week, 5060%
for transfers every two weeks, and 4050% for transfers every
three weeks. However, the timing of transfer of the explants from
callus induction medium to shoot induction medium did not have
any influence on the percentage of transformation. Approximately
the same percentage of transformation was obtained regardless
of whether the callus cultures were transferred to shoot induction
medium after four, six, or ten weeks on callus induction medium.
The data (Fig 1 ; Effect of transfer
interval on the percentage of explants transformed.) are representative
of this, shown here with a callus induction phase of four weeks.
THE EFFECT OF A LONGER CALLUS INDUCTION PHASE ON
SHOOT REGENERATION: No effect could be detected on the
percentage of explants transformed with different lengths of time
on callus induction medium before transfer to shoot induction
medium. By contrast, an effect could be observed on the number
of weeks before the appearance of the first callus with shoots
and the percentage of callus with shoots (Table 1).
The first callus with shoots was observed after 13 weeks on explants
exposed to four weeks callus induction medium. For explants exposed
to six weeks or ten weeks on callus induction medium the first
callus with shoots was observed after 28 and 34 weeks, respectively.
The percentage of callus with shoots for explants exposed to four
weeks callus induction medium was 82%. Whereas, the percentage
of callus with shoots was 6% and 1% after the same period for
explants exposed to six and ten weeks on callus induction medium
respectively.
Explants transferred to shoot induction medium after six or ten weeks might have resulted in a similar percentage of callus with shoots to those observed in explants transferred to shoot induction after four weeks, but the experiment was not carried on further than 35 weeks.
Table 1: The effect of a longer callus induction
phase on shoot regeneration
| Transfer to SI after 4 weeks | ||
| Transfer to SI after 6weeks | ||
| Transfer to SI after 10 weeks |
Sl: Shoot Induction medium
DISCUSSION AND CONCLUSION
The percentage of explants which were transformed, declined with
an increase in the time interval between transfers to fresh medium.
One reason could be that the explants of L. japonicus require
a very high level of cell division to give a high transformation
frequency. Although, 2,4D, which is a very potent inducer of callus,
is used during the callus induction phase, weekly transfers are
also required to give the necessary high levels of cell division.
This correlates with our unpublished observations that transformation
failed when 2,4D was replaced by NAA in the medium. Similar results
have been shown with other legumes, in which the presence of auxin
was shown to be necessary for transformation (De Kathen &
Jacobsen, 1994).
This experiment showed that a few weeks' delay in transfer to
shoot induction medium causes an even longer delay before the
appearance of the first callus with shoots and probably also reduces
the percentage of callus with shoots. Optimal production of shoots
in this system is only achieved when the tissue is transferred
at the right developmental stage. In transformed explants, this
implies that the timing of transfer is critical. One possible
explanation is that after a longer callus induction phase the
tissue would contain a high concentration of 2,4D. It would then
take a longer time for the plant hormone level to decrease sufficiently
to allow shoot development as 2,4D is not readily degraded by
the plant tissue.
We have shown that the transformation system for L. japonicus
is not very flexible when it comes to changing the regular
weekly transfers, but the transformation frequency is not affected
by the timing of the transfer of the explants to shoot induction
medium. Increasing the length of the callus induction phase did
influence both the timing of the appearance of the shoots and
the percentage of callus with shoots in the nontransformed
explants and a similar effect is expected for the transformed
explants. Therefore, to achieve the optimal shoot regeneration
the callus induction phase should be kept to a minimum.
REFERENCES
Andre De Kathen & HansJorg Jacobsen, 1994, Vlilth International Congress of Plant Tissue and Tissue and Cell Culture, S7104.
Kurt Handberg & Jens Staugaard, 1992, The Piant Journal 2(4), 487496.
Jennifer F Topping, Wenbin Wei & Keith Lindsey, 1 991, Development 1 12, 10091019.
K Judith Webb, Sue Mizen and Denise E Cooke
Institute of Grassland and Environmental Research
Aberystwyth, Dyfed SY23 3EB, UK
SUMMARY
During the six years of this study, GUS activity was more stable
in hairy root cultures than in either shoot cultures or plants
established in soil. The expression of the transgene in both shoot
cultures and the resulting whole plants was variable, particularly
in line 12, which contained multiple doses of the transgene.
INTRODUCTION
Lotus corniculatus is readily transformable with Agrobacterium
rhizogenes and has been used to investigate transformation
strategies in legumes. For example, both hairy root cultures and
regenerated transformed plants have been used to study primary
(Force et al., 1989) and secondary metabolism (Carron et
al., 1994) and activity of nodule specific genes (Jensen
et al., 1986). Longterm studies such as these highlight
the importance of the stability of expression of introduced genes
in hairy root cultures and their regenerants over an extended
period of time.
Two hairy root lines, 6 and 12, of L. corniculatus were
initiated in October 1988 and used to investigate the expression
of a tranegene throughout the transformed plants and in their
progeny (Webb et al., 1994). The easily identifiable reporter
gene uid, which encodes ß-glucuronidase (GUS),
was chosen for these studies. In addition, the stability of GUS
expression was studied in the root cultures themselves under a
range of conditions likely to be encountered in experiments (Cooke
and Webb, submitted). Here, we describe GUS activity in these
two root culture lines over a period of 6 years and in two sets
of transgenic plants regenerated during this growth period.
MATERIALS AND METHODS
The production and maintenance of the two root lines, shoots and
plants and their genetical, biochemical and molecular analyses
are described elsewhere (Webb et al ., 1994). These studies
showed that line 6 had one dose of the uid gene while line
12 had two or more independently segregating doses of the gene.
Both lines 6 and 12 contained multiple copies of the bacterial
TLDNA, while only line 6 was TR positive.
Data from enzymatic assays of GUS activity in lines 6 and 12 from
controls of four separate experiments, performed 3, 17, 32 and
55 months after culture initiation, are presented here. Root cultures
were grown in liquid culture (Webb et al., 1994) and harvested
710 days after subculture. The data are averages of at least
three replicate samples.
Stock cultures of hairy roots were routinely stored on agar plates
at 24°C in the dark, with regular subculture every
6 months. These cultures were maintained at 25°C in the dark
prior to establishing root cultures in liquid at 25°C at
10 µmoles m-2sec-1.ubcultured every
two weeks for experimental work. By contrast, shoots were excised
from 'old' root cultures maintained in the same liquid medium
for about 2 months. These shoots were maintained on semisolid
medium in tubes (Webb et al., 1994) at 20°C at 100 µmoles
m-2sec-1 subcultured every 4 months.
Shoots and plants were regenerated and established from both lines
of hairy roots grown in liquid at two different time intervals:
6 and 30 months (sets 1 and 2 respectively). The information presented
here is from plants in set 2; plants from set 1 were analysed
for GUS activity 14 months after initiation of the root cultures
(Webb et al., 1994). Shoots and plants in set 2 were excised from
hairy roots after 30 months and maintained as shoot cultures until
55 months. GUS activities were measured in: 1) leaves of different
ages in 6 or 7 separate shoot cultures and 2) leaves and roots
of surviving shoots after transfer to soil.
RESULTS AND DISCUSSION
GUS activity was detected in hairy root culture lines 6 and 12
over the entire growth period of 55 months (Fig. 1;
GUS activity in L. corniculatus hairy root lines 6 and
12 over 55 months). The greater GUS activity seen at 17 months
was probably due to differences in the sampling procedure. GUS
activity of control tissues was 0.063 µmoles MU µg protein-1
min.-1 GUS activity in line 6 was consistently lower
than that in line 12. This reflects the finding that GUS activity
in a variety of tissues, including roots, nodules, stems, leaves
and flowers, of the primary transformants (set 1) was lower in
line 6 than in 12 (Webb et al., 1994).
Shoot cultures of line 12 were routinely used as GUS positive controls in other transformation experiments. Loss of this activity was noted 50 months from initiation of the hairy root cultures Therefore, GUS activity in different tissues of lines 6 and 12 was measured. The results are summarised in Table 1. 29
Table 1: Summary of GUS activity in separate root and shoot cultures
of L . corniculatus after 55 month from initiation of hairy
root lines 6 and 12.
| Culture | Line 6 | Line 12 |
| Root cultures * | 6/6 (100%) | 6/6 (100%) |
| Shoot cultures ** | 5/6 (83%) | 1/7 (14%) |
| Plants in soil shoots | 4/4 (100%) | 0/5 (0%) |
| 0/4 (0%) | 0/5 (0%) |
* see Figure 1 for actual GUS activity
** shoots and plants from set 2
Key: 1 see Figure 1 for actual GUS activity
2 shoots and plants from set 2
All root cultures of both lines 6 and 12 were GUS positive and
were still positive 6 years (November 1994) after initiation.
Storage of the root cultures in the cold may have helped preserve
GUS activity in these lines.
In line 6, most of the shoot cultures were GUS positive, as were
the shoots of the four established transgenic plants; only the
roots and nodules of these plants were GUS negative. By contrast,
only 14% of the shoot cultures of line 12 expressed GUS. Neither
the shoot nor root system of any of the five plants of this line
had significant levels of GUS activity. Thus, plants established
in soil reflected GUS activity in the original shoot cultures.
The failure to detect GUS activity in the roots of plants of line
6 and a complete loss of expression in line 12 suggests that differentiation
of shoots and rooting of plants influenced expression of the transgene
in these two lines.
Various factors are known to affect transgene expression in primary
transformants, including site of insertion, copy number of the
transpene and methylation. One possibility is that these differentiated
tissues were more susceptible to methylation than their hairy
root counterparts.
REFERENCES
Carron, T. R., Robbins, M. P., Morris, P. (1994) Genetic modification
of condensed tannin biosynthesis in Lotus corniculatus. Heterologous
antisense dihydroflavonol reductase downregulates tannin
accumulation in 'hairy root' cultures. Theoretical Applied Genetics
87 10061015.
Cooke DE & KJ Webb (submitted) The stability of CaMV 35Sgus
gene expression in hairy root cultures of Lotus corniculatus
L. under different environmental regimes. Plant Cell, Tissue
and Organ Culture.
Forde, B.G., Day, H.M., Turton, J.F., Wenjun, S., Cullimore, J.V.
& Oliver, J.E. (1989) Two glutamine synthetase genes from
Phaseolus vulgaris L. display contrasting developmental
and spatial patterns of expression in transgenic Lotus corniculatus
plants. The Plant Cell 1 391401.
Jensen J.S., Marcker, K.A., Otten, L. & Schell, J. (1986)
Nodulespecific expression of a chimaeric soybean leghaemoglobin
gene in transgenic Lotus corniculatus. Nature 321 669674.
Webb KJ, MP Robbins & S Mizen (1994) Expression of GUS in primary transformants and segregation patterns of GUS, T, and TMDNA in the T, generation of hairy root transformants of Lotus corniculatus. Transgenic Research 3 232240.
F. Olmos
INIA Tacuarembo
The more important legumes in the northeast region of Uruguay are Lotus corniculatus and Trifolium repens (Allegri and Formoso, 1980; Olmos, 1991). When the soil is cultivated both produce 610 tons of dry matter/ha/year, but with a different seasonal pattern (Table 1) (Formoso and Allegri, 1983).
Table 1 - Seasonal dry matter production (%) of Lotus corniculatus
and Trifolium repens.
| Autumn | Winter | Spring | Summer | |
| T. repens | 22 | 23 | 52 | 3 |
| Lotus | 24 | 10 | 44 | 22 |
The rate of phosphate applied annually determines which of them
dominates in a pasture, when they have been sown together; the
higher rates give Trifolium repens pastures, while with
lower rates, white clover is lost and Lotus is still present
(Moron et al., 1982).
Besides this, climate factors affect the proportion of Lotus and T. repens on mixed pastures (Table 2). Dry summers increase the Lotus content in the next season, while the reverse is true if the precipitation matches the evaporation rate (Olmos, 1994).
Table 2 - Botanical composition of mixed pastures (%) in Spring
and Autumn-Winter.
In extensive grazing areas animal performance is limited by the
quantity and quality of the forage consumed, which is about 70
% of C4 grasses (Olmos and Godron, 1990).
Owing to economics and conservation factors Lotus and T.
repens have been introduced in the natural grasslands by oversowing.
The methodology works well for Lotus, but it fails many
times when used with T. repens.
In 1992 we started a set of experiments to assess the more important
variables (rate and time of sowing, sources and rate of phosphate
fertilizer) affecting Lotus establishment, productivity
and persistence with this method.
The quantity of phosphate applied was the most important variable, increasing LAI, forage quality, seed production and recruitment of new plants in the following season.
A simple matrix model was developed to study populations dynamics,
and showed that the persistence of the improved pasture relies
more on the recruitment of new individuals each year than on the
individual plant longevity.
BIBLIOGRAPHY
Allegri M., and F. Formoso. 1980 - Forage legumes in the northeast
region. CIAAB North Exp. Sta. Misc. 21.
Formoso F., and M. Allegri. 1983 - Forage production in Caraguata.
In: 1st. Regional Meeting on
Agric. Systems. CIAAB North Exp. Sta.
Moron et al. 1982 - Forage production with different phosphate
sources in a basaltic soil. In: Phosphate
sources for pastures. CIAAB Estanzuela Exp.Sta. Misc. no. 42.
Olmos F. 1991 - Cultivated pastures for the northeast region.
INIA Tech. Ser. no. 20.
Olmos F. 1994 - The effect of water deficits on the botanical
composition of cultivated pastures. INIA (in press).
Olmos F., and M. Godron. 1990 - Phytoecological survey in the northeast region. In: 2nd. National Meeting on Grasslands. Ed. Hem. Sur.
Lernmi G. and Negri V.
Istituto di Miglioramento Genetico Vegetale
Facoltk di Agraria, Universitk degli Studi
Borgo XX Giugno 74, 06100 Perugia (Italy).
INTRODUCTION
Lotus tenuis (2n=2x=12) can be crossed to L. corniculatus
(2n=2x=24) in seminatural conditions; the cross results in
a high fertile, tetraploid progeny morphologically resembling
birdsfoot trefoil. This suggests that the former species should
have contributed to the L. corniculatus gene pool through
unreduced (2n) gametes (Negri and Veronesi, 1989). Screening the
frequency of big pollen production in twelve natural populations
of L. tenuis (Negri, 1992), several 2n gamete producing
genotypes were found (Table 1). Crosses among 2x (L. tenuis)
x 4x (L. corniculatus) detected a 2n female gamete
producer (1770/16).
Table 1: Interesting populations, frequency of plants producing
more than 1% of big pollen in initial population, plants showing
the highest percentage of big pollen and their percentage of big
pollen production.
| Abbadia S Salvatore | |||
| Roseto degli Abruzzi | |||
| Ferro Monte Urano | |||
| Ancona | |||
| Monte Franco | |||
CYTOLOGICAL ANALYSIS
Cytological analysis revealed that different mechanisms are involved
in big pollen production. In two mutants (1321/8 and 1321/46)
parallel and bipolar spindles in metaphases II were observed.
As a consequence of parallel spindles, at the end of telophases
II, being the four sets of chromosomes localized in one plane,
dyads of 2n microspores were obtained. As for bipolar spindles,
after telophase II, two cleavage furrows was formed and a triad
of two n and one 2n microspores were obtained (Negri et al., 1994).
USE OF L. TENUIS MUTANTS IN TRASFERRING USEFUL CHARACTER
TO L. CORNICULATUS.
Since both the above mentioned mechanisms produce first division
restitution (FDR) type microspores, the examined genotypes are
presently used in transferring powdery mildew resistance and ability
to vegetate during the winter from L. tenuis to L. corniculatus.
In a first experiment 23 plants of the 1321/8 genotype and
9 plants of the 1321/46 genotype were planted under two isolation
cages with honey bees with a male sterile clone of L. corniculatus
(1766/81) for interspecific crosses. Seeds from the male sterile
plants were harvested separately; 9 mature tetraploids plants
(2 from 1766/81 x 1321/8 and 7 from 1766/81 x 1321/46), morphologically
resembling L. corniculatus, were obtained and cloned; parent
plants were also cloned. For powdery mildew resistance evaluation,
each plant within a clone was scored from 1=maximum resistance
to 9=minimum resistance (susceptibility) on August 8th, 1994 following
artificial inoculation as described in Veronesi et al. (1986).
For winter growth evaluation, plants were scored from 1=minimum
to 9=maximum growth on February the 2nd, 1994. Clonal evaluation
showed that only progenies from 1766/81 x 1321/46 have intermediate
characters (Table 2); among them two genotypes showed high resistance
to powdery mildew infection (x= 2.2 and 1.8, respectively) and
good winter growth (x= 6.1 and 7.3, respectively).
Table 2: Average values relative to clonal evaluation, of powdery mildew susceptibility
( 1= minimum, 9= maximum, August, 1994) and winter growth ( 1=
minimum, 9= maximum, February, 1994) in L. corniculatus female
parent, L. tenuis pollen parents and their progenies, ( in brackets
the number of clones evaluated)
| 1766/81x | ||||||
| 1321/8 (2) | ||||||
| Powdery mildew susceptibility | ||||||
| Winter growth | ||||||
SELECTION FOR INCREASING 2N GAMETE PRODUCTION
Pair hand crosses under isolation cages among nine 2n gamete producing
genotypes were conducted in 1993, in order to increase frequency
of 2n gametes production. We were not able to obtain an experimental
population with increased frequency of 2n gamete producing genotypes
since only 7 plants on 361 observed (2%), resulting from the crosses
1321/823 x 1321/828, 1321/828 x 1321/844
and 1170/73 x 1770/16, produced big pollen; but it is interesting
to note that these plants produced a much higher percentage of
big pollen (over 75%) than their parents (Table 1). Besides, 7
plants were found to be male sterile probably as a consequence
of accumulation of different mutations at different steps of the
meiotic process. Cytological analysis of mutants found is in progress.
Detection of 2n gametes producers might be influenced by variable
expressivity in relation to environment. Some clones of 2n pollen
producers are actually growing under two controlled environments
(20 hrs photoperiod and 20°C and 30°C, respectively),
to verify the effect of temperature on 2n gamete production.
REFERENCES
Negri, V., 1992: Frequency of big pollen occurrence in natural
populations of Lotus tenuis Wald. et Kit. In Mariani,
A. and S. Tavoletti (Eds), Proceedings of the Workshop: "Gametes
with somatic number in the evolution and breeding of polyploid
polysomic species: achievements and perspectives". Perugia
(Italy) 910 April 1992. pp.Sl54.
Negri, V. and F. Veronesi, 1989: Evidence for the existence of
2n gametes in Lotus tenuis Wald. et Kit. (2n=2x=12): their
relevance in evolution and breeding of Lotus corniculatus L.
(2n=4x=24) Theor. Appl. Genet. 78, 400404.
Negri, V., Lorenzetti, S. and G. Lemmi, 1994: Identification and
cytological analysis of 2n pollen producers in Lotus tenuis
Wald. et Kit. Plant Breeding. In press.
Veronesi, F., Negri, V. and A. Zazzerini (1986): Powdery mildew resistance in birdsfoot trefoil germplasm. Genetica Agraria 40: 387396.
Vignolio, O. R., N. O. Maceira and O.N. Fernandez
Ecologia, Unidad Integrada Balcarce FCAUNMdP/EEA INTA.
Balcarce, Argentina.
ABSTRACT
EFFECTS OF WATERLOGGING IN WINTER AND SUMMER ON THE GROWTH AND SURVIVAL OF LOTUS TENUIS AND LOTUS CORNICULATUS.
Tolerance to winter and summer waterlogging was experimentally
studied in Lotus tenuis and Lotus corniculatus. Both
legumes constitute an important forage resource in the Flooding
Pampa (Buenos Aires, Argentina), where L. tenuis occupies
environments more exposed to flooding than L. corniculatus.
Plants were cultivated individually in pots, which were kept
outdoors. Flooded plants were kept with a constant 3 cm water
level above the soil surface, while controls were periodically
watered. Plants were kept flooded until 75% of clorosis appeared
on either species (42 days in the winter treatment and 17 days
in the summer treatment. The winter treatment caused a decrease
in the aerial growth, leaf senescence, partial root decomposition
and the formation of shoot hypertrophies, but no mortality. L.
corniculatus was the most negatively affected species. Shoot
hypertrophies were more abundant in L. tenuis. Weight recuperation
after the winter waterlogging period was more rapid in L. tenuis
than in L. corniculatus. The summer treatment caused high
shoot senescence in both species and no hypertrophy formation.
After the waterlogging period, 50% of L. tenuis and 100%
of L. corniculatus plants died. Regrowth of surviving L.
tenuis plants was slow. The higher tolerance of L. tenuis
to waterlogging agrees with the habitat segregation of both
species, which has been in field studies.
Published: Ecologia Austral, 1994, 4: 1928.
A. D. Bavage and M. P.Robbins .
Cell Manipulation Group
Institute of Grassland and Environmental Research
Aberystwyth Research Center, Plas Gogerddan
Aberystwyth, Dyfed, U.K.
One aim of this group is to manipulate tannin biosynthesis in
forage legumes, by the use of molecular genetic techniques. A
key step in the biosynthetic pathway culminating in the production
of condensed tannin, is the reduction of dihydroquercetin and
dihydromyricetin by dihydroflavanol4reductase (DFR).
Using Agrobacterium rhizogenes mediated transformation
it has been possible to introduce antisense DFRgene constructs
into L. corniculatus (Carron, Robbins and Morris 1994).
Whilst it has been possible to monitor the expression of the introduced
heterologous antisense gene, analysis of the expression of the
native gene has proved difficult.
We report the use of the polymerase chain reaction to amplify
a fragment from genomic L. corniculatus DNA which corresponds
to part of a native DFR gene.
MATERIALS AND METHODS
Genomic DNA from L. corniculatus lines S33 and S50 (Carron,
Robbins and Morris 1994) was isolated as described by Robbins
et al., 1991.
Degenerate primers for PCR were designed based on the known sequences of DFR protein from Antirrhinum majus, Petunia hybrida and Gerbera hybrida. The 5' primer was a modification of that used by Helariutta et al., (1993) and comprised: AGAATGAAGT(G/T/A)AT(A/C/T)AA(A/G)CC (Primer 1).
Two 3' Primers were employed whose sequences were:
GGGTCGAC(A/G)CA(G/A/T/C)A(A/G)(A/G)TC(A/G)TC(G/A/T/C)A(A/G)(A/G)TG or
GGGTCTACCAT(A/G)TC(C/T)TC(G/A/T/C)A(A/G)(G/A/T/C)GT(A/T)TA
(Primers 2 & 3).The latter being located nearest to the 3' end of the gene.
Conditions for PCR product generation were optimized using a Petunia hybrida DFR cDNA clone (Clone pTIP1 supplied by J.Kooter).
Each reaction contained in a volume of 50µ1:
5µ1 100mM TrisHC1 pH8.5
5µ1 500mM KC1
5µ1 lmg/ml Gelatin
2µ1 50mM MgCl2
1µ1 10mM deoxynucleotides
0.5µ1 15µM Primer 1
0.5µ1 15µM Primer 2 or 3
0.2µ1 AmpliTaq DNA polymerase (1 unit)
10µl Target DNA: 1050ng Plasmid DNA or
100300ng Lotus genomic DNA.
Reactions were run in 0.5ml microcentrifuge tubes with the reaction mixture overlaid with 50~1 liquid paraffin. DNA was always made up in sterile distilled water. Liquid transfers were carried out using Aerosol Resistant Tips. All manipulations were conducted in a clean class 2 flow cabinet to reduce the risk of contamination from outside sources.
The following cycling conditions were used on a PerkinElmer 480 thermal cycler:
Hot start denaturation of: 94°C 3 minutes, 35 cycles of: 94°C 30 seconds, 55°c 1 minute, 72°C 2 minutes. Final extension of: 72°C 10 minutes. A 25,µl aliquot was taken from each reaction and run on a 0.5% agarose gel and visualized with ethidium bromide. When a product was detected a 1µ1 aliquot of the reaction mixture was used as a target for a second round of amplification using both primer 1/2 and primer 1/3 combinations. After photography gels were blotted onto HybondN membrane (Amersham) and then probed with an Antirrhinum majus DFR cDNA clone (Clone pJAM212Cathie Martin).
PCR products were cloned as follows:
A 25µ1 aliquot (l00ng1,µg product)from the reaction
mixture was precipitated by adding 0.1 volumes 3M sodium acetate
pH 5.2, 2 volumes ethanol and incubating on ice for 1 hour. The
DNA was precipitated by centrifugation 12000g for 15 min in a
microfuge. The supernatant was removed and the pellet washed with
200µl 70% ethanol (precooled 20°C). After
centrifugation for 5 minutes the pellet was air dried until all
traces of ethanol had evaporated. The pellet was resuspended in
50µ1 lx onephorall buffer plus (Pharmacia). The
ends of the fragments were blunted by the addition of l0µl
dNTP solution (2mM dATP, 2mM dCTP, 2mM dGTP, 2mM dTTP, Pharmacia)
and 2µl (2 units) T4 DNA polymerase (BoehringerMannheim).
The reagents were gently mixed and incubated at 12°C for
30 minutes.
An aliquot of 100500ng blunt ended PCR product was mixed
with 200ng Sma1 digested pUC18 and ethano1 precipitated
as described above. The pelleted DNA was resuspended in 7.0µl
water, 1µl l0x T4 DNA ligase buffer and 2µl (2 units)
T4 DNA ligase (GibcoBRL) and incubated 12°C overnight.
Half of the ligation mixture was used to transform CaCl2
competent E. coli strain DH5.
RESULTS
Using primers 1 and 2 a fragment of approximately 750bp was amplified
from both S33 and S50 genomic DNAs. With primers 1 and 3 a fragment
of approximately 1.5kb was generated (Fig).
Both of these fragments were around the predicted size for DFR
gene products assuming that the introns in the L. corniculatus
gene were similar to those of the published Antirrhinum
majus and Arabidopsis thaliana sequences.
When the large fragment from primers 1 and 3 was used as a template
for reamplification with primers 1 and 2 a 750 bp fragment
was produced. The 750bp fragment produced in both primary and
reamplification reactions crosshybridized with the
A. majus DFR probe (Fig).
The 750bp fragment from S50 was cloned into pUC18. Sequence analysis
revealed homologies between 71.6% and 68.3% over a 110bp overlap
with the A. majus, Arabidopsis thaliana, Hordeum vulgare, Petunia
hybrida, Vitis vinifera and Zea mays DFR PNA sequences
in the Genembl database.
DISCUSSION
Amplifications using degenerate primers for DFR initially produced
a series of fragments from L. corniculatus genomic DNA
(data not shown). After optimization of the reaction conditions
a single product was obtained with each pair of primers. This
product was isolated and shows homology to a DFR gene from A.
majus both by cross hybridization and sequence analysis. To
the authors knowledge this is the first tannin biosynthesis gene
fragment to be cloned from a Lotus species. The isolation of this
fragment should enable the entire gene to be isolated more easily.
The partial DFR gene clone will be useful in investigating the
expression of the native gene in L. corniculatus, both
in wildtype lines and transgenic lines harboring heterologous
DFR gene constructs.
ACKNOWLEDGMENTS
Thanks to Andrew Bettany and Kathryn Bradley for helpful advice
on PCR. To Tom Carron who designed primers 2 and 3. To Steven
Colliver who isolated the genomic DNA from L .corniculatus
and Mark Coleman at the University of East Anglia for the
PCR product cloning method.
REFERENCES
Carron.T.R.,M.P.Robbins and P.Morris. Genetic modification of
condensed tannin biosynthesis in Lotus corniculatus.1.
Heterologous antisense dihydroflavonol reductase downregulates
tannin accumulation in hairy root cultures. Theor.App.Genet. (1994)
87 p:10061015.
Helariutta.Y.,P.Elomaa,M.Kotilainen,P.Seppanen and T.H.Teeri.
Cloning of cDNA coding for dihydroflavonol4reductase
(DFR) and characterization of DFR expression in the corollas
of Gerbera hybrida var. Regina (Compositae). Plant Mol.
Biol. (1993) 22 p:183193.
Figure:
Analysis of PCR amplification products from Lotus corniculatus genomic DNA.
PCR products obtained from target DNAs: No target DNA, 2 P. hybrida DFR cDNA clone, 3 H. vulgare DFR cDNA clone, 4 L. corniculatus line S33 genomic DNA, 5 L. corniculatus line S50 genomic DNA, 6 product from primers 1&2 x 4, 7 product from primers 1&2 x 5, 8 product from primers 1&3 x 4, 9 product from primers 1&3 x 5.
M 100bp size marker ladder.
Cristina D. Strittmatter1; Rafael A. Ricco2
Mariana Kade1; Marcelo L. Wagner2; Alberto
A. Gurni2
1Centro de Ecofisiologia Vegetal., Buenos Aires, Argentina
2Catedra de Farmacobotanica. Fac. de Farmacia y Bioquimica.
UBA. Buenos Aires. Argentina
INTRODUCTION
Condensed tannins (flavolans) are the fourth most abundant plant constituent (Muthukumar et al., 1985). The aim of this study was to describe condensed tannins (CT) in Lotus tenuis, a nonbloating pasture legume naturalized in Argentina's most important region for calves production (Flooding Pampa).
Porter (1988) has reported the presence of proanthocyanidins (procyanidin and prodelphinidin) in the roots of the mentioned species. Estrella and Ugalde (1993) have not detected any anthocyanidins in the leaves of L. tenuis from the same geographic area where grew the exemplars analysed in the present study.
Fresh roots, stems and leaves were analysed to determine
whether or not CT were present, and their location in the different
plant tissues.
MATERIALS AND METHODS
Seeds of L. tenuis from the Flooding Pampa were germinated in the greenhouse. Plants were collected when buds were produced.
The samples for histological observations were obtained by cutting fresh leaves, stems and roots into thin transversal sections. The roots were cut at different levels, including the nodules.
The reaction with vanillinHCl was performed on all the slices, before the observation under microscope at l0X and 40X.
Characteristically, a cherry red colour is produced in presence
of proanthocyanidins after treatment with the mentioned reactive
(Sarkar and Howarth, 1976).
RESULTS
Distribution of condensed tannins in Lotus tenuis
| Leaves | ||
| Stems (Fig.1) | Pith: few isolated | |
| tanniniferous cells. (Fig.2). | ||
| Roots (Fig.3) | Cortex: few isolated | |
| tanniniferous cells. | ||
| Roots at nodule level | ||
| -Nodules (Fig. 4) | Cortex: external periferical zone | |
| forming a continuous band | ||
| Pith : diffuse reaction. | ||
| Roots (Fig.5) | Cortex: high concentration in | |
| the whole zone. |
- : no detected; (+) :traces; = : presence; ++ : abundance
CONCLUSION
The biological role of CT in the roots seems to be related to nodulation (Estrella and Ugalde, 1993). The slices of roots at the same level than the nodules show an intensive reaction with vanillinHCI, which indicates a possible response of the roots to the infection with Rhizobium loti present in the nodules.
The concentration of CT in roots and stems of L. tenuis is
very low as to be detected by means of the usually employed
phytochemical procedures, but the species is capable of synthetising
them. The virtual lack of CT in L. tenuis provides a mean
in order to distinguish the species from others which produce
these compounds in higher concentrations (Estrella and Ugalde,
1993). From this point of view, the CT could be employed as systematic
markers within the genus.
REFERENCES
Estrella, J.M. and Ugalde, R.A. (1993). Analisis de los flavolanos
en especies del genero Lotus y su efecto sobre el crecimiento
in vitro de Rhiizobium loti. Actas XX Reunion Argentina
de Fisiologia Vegetal. pp. 326327.
Porter, LJ. (1988). Flavans and Proanthocyanidins. In Harborne,
J.B. The Flavonoids. Advances in Research Since 1980. Chapman
and Hall Ltd. London New York pp. 2162.
Muthukumar, G., Sivaramakrishnan, R. and Mahadevan, A. (1985).
Effect of tannins on plants and their productivity. Proc. Indian
Natl. Sci. Acad. Part B Biol. Sci. 51 (2): 270281.
Sarkar, S.K. and Howarth, R.E. (1976). Specificity of the vanillin test for flavanols. J. Agric. Food Chem. 24 (2) : 317320.
D. R. Viands1, N. J. Ehlke2, Y. A. Papadopoulos3,
and R. R. Smith4
1Cornell Univ., Ithaca, NY
2Univ. of Minnesota, St. Paul, MN
3Agriculture Canada, Nappan, Nova Scotia
4U.S. Dairy Forage Research Lab., Madison, WI.
During the 1993 technical committee meeting of the NE144
Regional Cooperative Research Project, "Forage Crop Breeding
to Improve Yield and Stability", breeders indicated that
specific pathogens recently were identified in different areas
of North America that reduce productivity and stand life of birdsfoot
trefoil. Because of limited resources for breeding birdsfoot trefoil,
each breeder is not able to embark on a new breeding program for
every disease resistance. Therefore, plans were developed this
past year to cooperate in breeding birdsfoot trefoil with multiple
disease resistance.
The table below lists the cooperators and the pathogen(s) isolated
from each location. Because of apparent plant genotype X Fusarium
oxysporum isolate interaction for disease severity, the isolates
from NY and WI will be treated separately in the selection programs.
| Location | Breeder | Pathologist | Pathogens |
| Minnesota | N.J.Ehlke | S. Samac | Fusarium acuminatum, F. equisiti |
| Wisconsin | R.R. Smith | C. R. Grau | F. oxysporum |
| New York | D.R. Viands | G.C. Bergstorm | F. oxysporum |
| Nova Scotia | Y.A. Papadopoulos | J. Kimpinski | Pratylenchus penetrans |
Each cooperator will conduct recurrent phenotypic selection for resistance to the disease identified at his/her location. Selection will be done within a source population from each of the other cooperators as well as his/her own. Source populations will be kept separate until three to four cycles of recurrent selection are complete. At the completion of selection, the following is proposed:
1. Determine progress from selection for resistance to each of the diseases.
2. Evaluate the impact resistance makes on productivity and persistence at various field locations.
3. Return selected subpopulations to the breeder from which they
were derived. Each breeder has the option of pooling subpopulations
derived from his/her own source population. Pooling subpopulations
probably will result in a population with moderate levels of resistance
to all these diseases. If desired, further selection may increase
the level of resistance.
We hope this research will result in birdsfoot trefoil germplasm with resistance to many of the major diseases that limit production within northern USA and Canada. This cooperative effort is necessary where breeders are able to devote only a small proportion of their total effort on this crop. Collectively, significant impact is anticipated in developing birdsfoot trefoil that will maintain broad adaptation.
W. F. Grant1 and I. Altosaar2
1Department of Plant Science, Macdonald Campus of McGill
University, Ste. Anne de Bellevue, Quebec, Canada
2Department of Biochemistry, University of Ottawa,
Ottawa, Ontario, Canada
The use of electrophoretic techniques in taxonomic and genetic
studies has been well established (Altosaar et al. 1974). Acrylamide
gel electrophoresis is a highly reproducible method involving
electrophoretic mobility as well as the molecular sieving
action of the gel that resolves plant proteins into many fractions.
The species used in this study are the diploids L. uliginosus
(B193) and L. tenuis (B109), a diploid
hybrid (L. burttii (B303) X L. alpinus (B77))
and the tetraploid L. corniculatus (B534).
EXPERIMENTAL PROCEDURES
Only young leaves (first, second and third leaves from the top) in identical developmental stages were used. Simple distilled water extracts in conjunction with dialysis concentration steps did not yield satisfactory results. The procedure of Nash (1968) with modifications produced extracts which showed the greatest number of clear bands upon fractionation by electrophoresis. All work was performed in a cold room at 6°C.
Five grams fresh weight of leaves were washed in a 9 cm plastic
Petri dish with two 20 ml aliquots of distilled water and one
ml aliquot of extraction buffer (0.059 M trigphosphate,
pH 6.9). The leaves were then ground to a slurry in a prechilled
mortar, together with 10 ml of extractant. This slurry was poured
into a 30 ml Pyres clear glass homogenizer standing in an ice
bath; ten passes were made using a Teflon pestle attached to a
Fisher DynaMix. After homogenization, 2.5 g of equilibrated
Polyclar AT was allowed to equilibrate with the extractant for
24 h. Excess extractant was removed by centrifugation at 325 X
g for 5 min before adding the moist Polyclar to the tissue homogenate.
After standing for 10 min, the Polyclarhomogenate mixture
was filtered through 4ply cheesecloth and the filtrate centrifuged
at 15,000 X G for 20 min in a Sorvall Superspeed centrifuge at
0°C. The clear supernatant was concentrated three to
fourfold with two 30 min concentration steps in dry G25
Sephadex according to Nash (1968). Protein concentrations were
determined using the Waddell formula: 1lgm/ml of protein = (OD
at 215 mp OD mp) X 144 (Nash 1968). Scanning was done in
a Unicam spectrophotometer. The supernatant was adjusted accordingly
with extractant to yield a 200 1lgm/ml solution when diluted 1:1
with 0.059 M trigphosphate buffer containing 50% sucrose.
Preliminary studies in which the amount of protein applied on
each gel varied from 150 gm/ml to 525 gm/ml, indicated that the
clearest patterns were obtained from 200 gm/ml concentrations.
Disc electrophoresis was carried out. The spacer gel, containing 0.47 M trisphosphate buffer, pH 6.0, was polymerized over the separation gel and did not contain sucrose. Electrophoresis was conducted at 6°C in rectangular buffer tanks for 25 min at 4 mamp/gel and subsequently for 90 min at 6 mamp/gel. Gels were stained in 1% Aniline Blue Black in 7.5% acetic acid for at least 30 min and differentiated in 7.5% acetic acid.
The Rf values for each band were calculated from average
data obtained from 4 or 5 electrophoretic runs.
RESULTS AND DISCUSSION
Leaf extracts of each of the taxa displayed from 6 to 15 relatively
single protein bands (Figs. 14).
In most cases, the bands were distributed almost the entire length
of the gels, and varied in intensity from faint and narrow to
dark and broad. Each electrophoretic run produced nearly identical
Rf values for each taxon, so the amount of intraspecific
variation was negligible, as long as similar ontogenetic stages
were used. Comparison among the four runs shows that the banding
pattern is a distinct specific characteristic. In the case of
L. uliginosus (Fig. 1) only six faint bands were detected
whereas for L. corniculatus (Fig 4) 15 bands were detected.
It is clear that the banding patter for L. uliginosus is quite
distinct from that for L. corniculatus as has been found
for isoenzyme data (Raelson and Grant 1988). The banding pattern
for L. tenuis (Fig. 2) while differing from that of L.
corniculatus is at the same time more similar to L.
corniculatus than to L. uliginosus. The banding pattern
for the hybrid L. burttii X L. alpinus (Fig. 3) again while
differing from L. corniculatus is more similar to L.
tenuis and L. corniculatus than to L. uliginosus.
It is clear from these results that L. uliginosus would
appear to be less related to L. corniculatus than L.
tenuis and L. burttii L. alpinus to L. corniculatus.
REFERENCES
Altosaar, I., Bohm, B. A. and Ornduff, R. 1974. Discelectrophoresis
of albumin and globulin fractions from dormant achenes of Lasthenia.
Biochem. System. Ecol. 2: 6772.
Nash, D. T. and Davies, M. E. 1972. Some aspects of growth and
metabolism of Paul's Scarlet rose cell suspensions. J. Exp. Bot.
23: 7591.
Raelson, J. V. and Grant, W. F. 1988. Evaluation of hypotheses concerning the origin of L. corniculatus using isoenzyme data. Theor. Appl. Genet. 76: 267276.
Nagy Laszlo
Irrigation Research Institute
Szarvas, Hungary
We presented accurate data of experiment founded in 1991
in connection with forage, seed yield and weeding facilities by
the result of 1991, 1992, 1993 years in the last year (see
Lotus Newsletter 1993 2325 p.).
In this year we tested yielding capacity, number of plant per
meter, weeding and not at last the germination capacity, 1st table.
By the mean results it could be expressed that:
stands broadcasted with the higher seed dosages have higher
seed yields, plant density, lower weeding and practically the
same germination than the population after the lower seed dosage,
the stands got different herbicide treatments shown the
auspicipous effect of imazetapir on the seed yield, weeding and
unfavorable effect on plant number and germination,
the mean data of cutting variant show that, cut done just
before flowering has much better effect on seed yield and germination
than the cut after flowering. But the plant density was better
in the event of post flowered situation.
The poorer result of seed yield and germination of stands cut
after flowering phase are first of all in connection with the
season of this year.
Table 1. The effect of seed dosage. Weed control from and the
phenophase of first cutting of the fourth year seed yield, plant
density, weeding and germination of bird's foot trefoil. Szarvas.
1994
