Selection for Chlorsulfuron Resistance in Flax (Lin urn usitatissirnurn) Cell Cultures MARK C. JORDAN and ALAN McHUGHEN Crop Development Centre, University of Saskatchewan, Saskatoon, Sask. S7N OWO Canada Received February 20, 1987 . Accepted April 17, 1987
Summary Seed transmission of novel traits selected from cell cultures needs to be established before somaclonal variation and cellular selection schemes can be applied to field crop improvement. Callus cultures were initiated from hypocotyl segments of oilseed flax (Linum usitatissimum) and transferred to selection medium containing chlorsulfuron , the active ingredient in the herbicide Glean*. After a suitable period, surviving colonies, or «green spots" of cells wece isolated from the selection medium and placed in regeneration medium, where shoot reorg-anization occurred in some cases. Whole. fertile plants were evemually recovered from several chlorsulfuron resistant cell lines. and their seeds were germinated and hypocotyls taken to initiate callus cultures. To test seed heritability of the chlorsulfuron resistance. this callus was subdivided and placed onto the selection medium and scored for survival. Callus from several progeny showed resistance to chlorsulfuron. indicating seed heritability of the novel trait .
Key words: Linum usitalissimum. flax. linseed, celluLar seLection, herbicide resistance. heritabiL· ity, chlorsu/furon.
Introduction Plant cells in vitro can undergo spontaneous genetic changes which can he manifest in the plants regenerated from the cells; this phenomenon has been termed somaclonal variation (Larkin and Scowcroft, 1981). If a trait of potential agronomic value can be created and identified in cells in culture, and those cells regenerated into plants whose progeny express the trait, it could save considerable time and effort in conventional beeding programs. Cellular selection procedures have been used to identify herbicide resistant cells in vitro, cells which subsequently regenerate into herbicide resistant plants. For example, Aviv and Galun (1977) regenerated propham resistant plants from selected cells, Miller and Hughes (1980) produced paraquat resistant plants, as did Thomas and Pratt (1982); picloram resistant plants were produced by Chaleff and Parsons (1978), glyphosate resistant tobacco was regenerated by Singer and McDaniel (1985), who earlier produced amitrole resistant plants (1984), and broad resistance to the imidazolinones in maize was created by Anderson and Georgeson (1986). H owever, seed transmission of the resistance character, which is necessary for the application of this technique to field crop improvement, has only been documented in a few cases, such as Chaleff and Parsons (1978), Thomas and Pratt (1982) and Anderson and Georgeson (1986). The herbicide Glean~ (chlarsulfuron) is registered far usc far pastemergent control of broadleaf weeds in wheat and barley. Due to low soil moisture and the presence of
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alkaline soils, the herbicide has a long residual effect in many agricultural areas. Up to 16 % of the applied chemical can be present in the following season (Smith and Hsiao, 1985), thus restricting crop rotation options. Flax (Linum usitatissimum) shoots and leaves can metabolize chlorsulfuron to nonphytotoxic compounds (Hutchison et aI., 1984) but the roots cannot. This means that flax can be sprayed post-emergent with chlorsulfuron, but cannot be seeded into soil containing chlorsulfuron either from fresh application or from residue from a previous season. The development of flax roots with increased resistance to chlorsulfuron would provide producers with greater crop rotation flexibility. Chlorsulfuron is an inhibitor of acetolactate synthase (ALS), an enzyme involved in the biosynthesis of branched chain amino acids (valine, isoleucine) (Chaleff and Mauvais, 1984). Because of this, resistance might be identified at a cellular level. Chaleff and Ray (1984) used a cellular selection system to identify chlorsulfuron resistanee in Nicotiana tabacum; the resistance trait was seed transmitted in a dominant or semidominant manner. Here, we document the use of a cellular selection system to identify flax cells resistant to the herbicide in vitro, regenerate plants and test progeny callus cultures for resistance.
Materials and Methods Flax [Linum usitatissimum L. cv. «McGregofll> and STS-2 (a line regenerated from cells selected for salt tolerance in vitro (McHughen and Swartz, 1984)] seeds were surface sterilized and germinated under aseptic conditions in a dark cabinet. Hypocotyl segments 1-2cm in length were isolated from 5 to 6-day-old seedlings and placed on Murashige and Skoog (1962) (MS) medium supplemented with 1 mg ·1- I 2,4-D, 0.8 % agar a'n d 3.0 % (w/v) sucrose in petri plates which were then sealed with Parafilm and placed in an incubator with a 16 h light period. After 4 weeks the resulting callus tissue was removed, cut into small ( -1- 2 mm 3) pieces and transferred to MS basal medium containing 100 or 200 nM chlorsulfuron. The chlorsulfuron was added to the media by the procedure of Ray (1984). Half of the calli from the 100 nM chlorsulfuron treatment were treated with the mutagen sodium azide by adding one drop of 0.1 M NaN, in phosphate buffer (PH 3) to the callus (Hibberd and Green, 1982). Although sodium azide had not previously been shown mutagenic to flax specifically, background experiments here have shown this level of sodium azide to inhibit regeneration from flax hypocotyl segments in vitro, indicating mutagenic activity (data not presented). Normal, healthy green callus can survive on MS basal medium for about 5 weeks before showing signs of necrosis (McHughen and Swartz, 1984). The stress imposed here by the chlorsulfuron begins quickly and after 4 weeks only occasional green spots remain among brown necrotic cells. Surviving green spots were isolated and transferred to MS medium with 1 mg .1- 1 6-benzyladenine (BA) to stimulate shoot formation, while residual portions of the green spot were cycled back onto selection medium with 200 nM chlorsulfuron. There was no chlorsulfuron in the regeneration medium. Shoots were transferred to MS basal medium in vials to facilitate rooting. Successfully rooted plandets were transferred to a mist chamber in a sterile sand-vermiculite mix (1: 1) for 3 -4 weeks before transplanting to regular soil in pots in the greenhouse, where the plants were permitted to mature, flower and set seed. The residual surviving callus was subjected to further cycles of exposure to MS with 200 nM chlorsulfuron for 4 weeks followed by 4 weeks in MS with 1 mg ·1 - I BA, with any regenerated. shoots transferred to vials as above. Seeds from putative chlorsulfuron resistant regenerants were germinated under aseptic conditions and callus produced from hypocotyl segments as in the initial steps. The resulting callus
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was placed on 100 nM chlorsulfuron (in MS medium), the level in the initial selection. Calli were scored for survival after 4 weeks.
Results and Discussion Selection on 100 nM chlorsulfuron yielded similar numbers of green spots for both the STS-2 and the McGregor (Table I). The sodium azide treatment resulted in a larger number of green spots from STS-l calli, but the difference was not pronounced_ Surviving green spots were isolated and transferred to regeneration medium (MS plus I mg .1- 1 BA). Regenerants GI-4, GI-9, GI-IO and GI-II arose during the first 4 week cycle on regeneration medium; GI-7 required two. Residual material from the selected spots was subjected to another selection phase, this time consisting of 4 weeks on MS medium with 200 nM chlorsulfuron followed by 4 weeks on MS with 1 mg ·1- 1 BA (regeneration medium). Regenerants GI-I, GI-2, GI-3, GI-S, GI-6 and GIS arose after the second cycle on the higher selection medium. Of the original 18 green spots of STS-2 from the 100 nM chlorsulfuron treatment, 14 survived the repeated cycles but only 5 of the original 13 McGregor green spots survived the repeated stress. When the original selection medium contained 200 nM chlorsulfuron, only the STS-2 callus yielded green spots (Table 1). Of these 10 isolated green spots,S survived 4 furrher selection cycles of 200 nM chlorsulfuron followed by MS basal medium. None, however, was successfully regenerated_
Table 1: Frequency of green surviving colonies on calli from two genotypes on three treatments [1DOnM (A), 100nM plus sodium azide (B), and 200nM chlorsulfuron (C) in the selection medium}_
----------------------------------------# Calli # Spots
% calli with spot
McGregor A B
C
STS-2 A
31 6 19.3
80 0 0
36 6 16.6
30 7
23.3
Total
B
C
36
72
33
10 13.9
12
252 41 16.3
Table 2: Survival of calli derived from seedling progeny of regenerants from chlorsulfuron selected cell lines, placed on MS basal medium with 100 nM chlorsulfuron added. See also Fig_ L
- - - - --_.
# progeny
Regenerant
selection cycles"')
GI- 4 Gl- 6 Gl- 7 Gl- 8 GI- 9 GI-IO GI-II
I 2
4
2
20 5
3 3
#
surviving calli
3 0 0 9 4 2 2
*) number of cycle!: of original surviving colony in selection medium.
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Fig. 1: Petri plate (60mm diam.) containing lOOnM chlorsulfuron in MS basal medium, inoculated with two calli from non-selected control material (at top) and callus pieces from the progeny hypocotyls from selectant GI-IO, after four weeks, Calli were of similar size at inoculation; the two control calli have died while the progeny of selectant GI-IO remain green and relatively healthy.
Cell lines have also been recovered which are resistant to 400 nM chloTSulfuron, but these lines have lost their regeneration capability) and therefore are of little agronomic value.
The amount of chlorsulfuron needed to effectively kill flax callus is much greater than that needed to kill tobacco callus. Chaleff and Ray (1984) used 5.6 nM chlorsulfuron for their selection level. It is not known if flax callus can metabolize chlorsulfuron and therefore needs a high dose to compensate and overcome the mechanism,
or if flax is more resistant to chlorsulfuron from high endogenous levels of ALS, or if flax ALS has a lower affinity for the chlorsulfuron. Flax root elongation is sensitive
to levels as low as 1 nM (data not shown), indicating that healthy green callus cells are metabolizing the chlorsulfuron.
The ability of STS-2 to generate more resistant colonies than McGregor (from which STS-2 was derived) might be due to the fact that STS-2 was selected from a cell line surviving a saline stress in vitro (McHughen and Swartz, 1984). In flax, responses
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to stress (including cells in vitro) are known to include rapid changes in repeated DNA sequences (Cullis and Cleary, 1985) which can persist over several generations. Thus the STS-2 line might differ from its progenitor (McGregor) in its composition of repetitive DNA, which could allow it to generate variability at a higher frequency than McGregor when exposed to stress. This variability might involve changes in the way the gene for acetolactate synthase or genes involved in detoxification are expressed. For example, overproduction of ALS or of a detoxification gene through increased transcriptional rates would lead to the resistant phenotype. Alternatively, amplification of certain repetitive DNA sequences could lead to simultaneous amplification of the above mentioned enzymes if the genes are located in close proximity to the repetitive sequences. The ability of STS-2 to survive levels of chlorsulfuron better than McGregor might be due to an increased vigor of the STS-2 line, which, in field situations, shows greater vigor than McGregor even under non-stressed soil conditions. Eleven plants were regenerated from McGregor cells treated with sodium azide and isolated as survivors from the lOa nM chlorsulfuron treatment. Four plants (Gl-I, GI2, GI-3, and GI-7) were regenerated from the same surviving colony while the other seven were from different spots. Anomalously, only the sodium azide treated surviving colonies of McGregor responded to the regeneration medium. Of the eleven plants, one died in the greenhouse prior to flowering, one was sterile and one much later to mature than the others in the same batch, so was not included in subsequent experiments. Later analysis showed that all progeny derived calli from this late maturing regenerant were chlorsulfuron resistant. The other seven original regenerants were similar to many flax primary regenerants in that they grew poorly and with reduced fertility and seed set, but producing larger than normal seeds. These seeds germinate very slowly and often have difficulty establishing in soil. Plandets grow slowly and tend to be late maturing. Attempts at crossing this material have been unsuccessful. Callus was derived from the putative chlorsulfuron resistant progeny seed. In every case the number of progeny seed available was too low for reliable analysis of resistance or genetic segregation in whole organisms, so hypocotyls were used to produce callus for testing in vitro. The callus was then tested for chlorsulfuron resistance to determine whether the trait was genetically stable and seed heritable. Table 2 shows the data and indicates that at least some of the selectants gave rise to progeny that segregated for the trait, at least at the cellular level, although the numbers are too low to be conclusive. Callus derived from hypocotyls from two seeds from regenerant GI-5 were sensitive to 100 nM chlorsulfuron, but also died on nonselection medium, so no conclusions can be drawn concerning chlorsulfuron resistance in this case. Rcgcncrant GI-B had 20 progeny (seeds); the callus from 9 of these were resistant and 11 were sensitive. Chaleff and Ray (1984) found that Nicotiana tabacum regenerants selected for resistance to sulfonylurea herbicides (such as chlorsulfuron) carried a single dominant (or semi-dominant) nuclear mutation conferring resistance, so a similar mutant here would be expected to produce a I: 2 : I progeny ratio (from a ]. Plan! P/rysiol. Vol. 131. pp. 333-338 {1987}
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selfed heterozygote), with a 3: 1 resistant to sensitive phenorype. Regenerant GI-8, the only one with enough seeds for analysis, shows a ratio like 1 : I. Further analysis with larger numbers of samples will help to conclusively determine the genetic and physiological nature of the trait. We have shown that it is possible to generate and select chlorsulfuron resistant cell lines of flax, to regenerate plants from the selectants and show resistance in the progeny. The lines ;rre presently being increased to provide sufficient numbers to test under field conditions at the whole plant level, and also to analyse the acetolactate synthase properties. References AVIV,
D. and E. GALUN: Isolation of tobacco protoplasts in the presence of isopropyl N-phenyl-
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