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Cucurbit Genetics Cooperative Report 15:97-101 (article 37) 1992

Some considerations about expression of marker genes in experiments of genetic transformation in Cucumis anguria var longipes by coculturing cotyledon explants with Agrobacterium tumefaciens

R. Santana, V. Moreno and L.A. Roig

Plant Cell & Tissue Culture Laboratory. Biotechnology Department, Universidad Politecnica de Valencia. Camino de Vera, 14. 46022-Valencia (Spain)

Introduction

Cucumis anguria var longipes is a wild cucurbitaceous species in which resistance to 'yellowing virus disease', cucumber green mottle mosaic virus, powdery mildew (Erisiphe cichoracearum DC ex Merat), nematodes (Meloydogine sp.) and bean spider mite (Tetranychus urticae Koch.) have been described (5, 9, 10, 11; for a revision see 14). The introduction of these genes in the genetic background of melon (Cucumis melo L.) would represent an important improvement of this crop species. Since for some of these plagues or diseases there are not sources of resistance within the species and because of the existence of incompatibility barriers for the sexual cross with the wild species, the most feasible alternative is the somatic hybridization by protoplast fusion. But for the efficient application of this technique it is necessary to have a good method for the selection of the somatic hybrids among the parental plants. So, the use of Agrobacterium tumefaciens strains as a biological vector for introducing marker genes in the wild species would facilitate that selection.

In this work, the results of a preliminary study about the infectivity of a bacterial strain (LB4404) carrying the binary plasmid pBl121 with two marker genes (β-D glucuronidase and neomycin phosphotransferase II) and the attainment of several Cucumis anguria plants expressing both markers, as described.

Methods

Plant material. Seeds of cucumis anguria var longipes L-2 (accession No. GBNR 1751, kindly supplied by Dr. Den Nijs, I.V.T., Holland) were decoated and surface sterilized by immersion in 50% commercial bleach (50 g/l active chlorine) for 25 min, followed by three rinses with sterile distilled water. Germination was carried out in MG medium (15) under photoperiod (16 h light, cool fluorescent tubes,m Gro-lux, Sylvania, 90 μ E seg-1 m-2), at 27 ˚ C. Cotyledon from these plantlets on the appearance of the first true leaf were used as explant source as previously described for melon (13).

Agrobacterium tumefaciens Strain LBA4404 (kindly supplied by Dr. A. Granell, IATA, Valencia, Spain) carries rifampicn resistance in their genophore and the pBl121 binary plasmid (8), a pBIN19 (1) derivative, with two genetic markers of bacterial origin not found in plants: that of β -D glucuronidase(GUS, EC 3.2.1.31) a hydrolase active against a wide number of β -glucuronides, which under the control of the CaMV35S promoter is able to express in plants, and that of neomycin phosphotransferase II (NPTII, from transposon Tn5) which inhibits the action of the aminoglycoside antibiotics by phosphorilation and, under the nos promoter, confers resistance to kanamycin in plants. Bacterial growth was carried out in Luria Broth (12) supplemented with 50 mg/l rifampicn (Rifaldin, Merrell Dow Pharm. Inc.) and 100 mg/l kanamycin (Sigma) at 28 ˚ C in darkness, overnight, in an orbital shaker at 250 rpm.

Sensitivity of explants to kanamycin. In this work, the culture media and incubation conditions applied to the explants in order to obtain plant regeneration from them were the most suitable chosen from a previous study (data not shown) that summarized as follows:

A. - Organogenic medium: NB 0.1/1.0 Cu CM consisting of basal medium (13) supplemented with 0.1 mg/l NAA (naphtalene acetic acid), 1.0 mg/l 6BA (6-benzyl-adenine), 1.0 mg/l SO4Cu, 10% (v/v) coconut milk, and 20 g/l sucrose.

B. - Shoot-developing medium: the organogenic zones appeared in the preceding medium were excised from the callus and subcultured in the shoot-developing medium MB 0.01/0.1 Cu (14).

C. - Rooting medium: well developed shoots were rooted in medium MB3 (15).

To determine the suitable dose of kanamycin for the selection of transgenic plants this antibiotic was added to the explant morphogenetic culture medium at four concentrations: 100, 200, 500 and 750 mg/l. Incubation was done in the same growth chamber already described for seed germination.

Variables measured were callus growth and organogenic response, expressed as percentage (+ SE) of explants giving unorganized growth and organogenic structures, respectively, with readings taken after 21 and 28 days of culture.

Transformation. In order to check if an increase in the initial number of potentially transformable cells produced an increase in the transformation efficiency, two kinds of cotyledon explants were used in this experiment: those directly taken from the plantlets and those precultured in a callus promoter medium (NB 2.5/1.0) consisting of basal medium supplemented with 2.5 mg/l NAA, 1.0 mg/l 6BA, 20 g/l sucrose and 8 g/l agar (Technical No. 3, Oxoid). 200 explants were used for bacterial transformation and 60 for control in both kinds of explants.

Infection of the explants was made in a relatively short time (~ 7 min.) with a mixture 1:1 (v/v) of the morphogeneic liquid medium NB 0.1/1.0 Cu CM and an overnight culture of Agrobacterium. After infection, explants were dried off with sterile filter paper and cocultured onto solid morphogenetic medium for 2 days at 28 ˚ C in darkness/ After coculture the explants were taken out of the medium, washed in M&S salt solution containing 500 mg/l cefotaxime (Claforam, Roussel) to remove the bacteria, dried on filter paper and put onto selective solid morphogenetic medium containing 100 mg/l kanamycin as the selective agent and 300 mg/l cefotaxime to prevent the bacterial growth.

After 7 days in this medium (to check the no apparition of bacterial growth and the absence of necrosis in the explants) they were transferred every 14 days to fresh medium to permit the calus growth. Morphogenetic calli grown in this medium were selected and transferred to shoot developing medium NB 0.01/0.1 Cu maintaining the selective agent (100 mg/l kanamycin) and the bacterial controlling antibiotic (300 mg/l cefotaxime). Later subcultures on this medium without the selective agent were also carried out to check its influence on the develoment of shoots. Shoots were transferred to basal medium with 300 mg/l cefotaxime for rooting.

Test for GUS expression. Plants regenerated from organogenic calli grown with kanamycin and also developed in the selective medium were analyzed for the expression of GUS gene according to a modification of Jefferson's procedures as follows: 10 mg pieces of leaves were lysed and homogenized with 100 μl extraction buffer (50 mM sodium phosphate, 10 mM EDTA-Na, 0.1% Triton X-100 and 10 mM β -mercaptoethanol, pH 7.0) in ice-cooled baby Eppendorf. Samples of 40 μl were incubated with 60 μl substratum (1 mM 4-methyl-umelliferyl- β -glucuronide, Sigma, inextraction buffer) at 37 ˚ C in darkness for 20 min. Fluorescence was observed with a transilluminator under long wave UV light.

Results

  1. Sensitivity to kanamycin Experiments about kanamycin sensitivity of cotyledon explants of Cucumis anguria var longipes indicated that a concentration of 100 mg/l was selective. The initial growth, although in the 31% of the explants, was extremely low (almost nil) and it was finally inhibited by the antibiotic. The morphogenetic response was nil from the beginning and was not observed either after 28 days in culture. Concentrations of 200 mg/l and higher were absolutely deleterious for growing the explants.
  2. Transformation. Since the explants (both direct and precultured) did not present bacterial growth or necrosis, on day 7 they were transferred to fresh medium for an additional 14 days, and subcultured again for another 14 days. The frequencies of calli with organogenic response grown from those explants are shown in Table 1.

Table 1. Pecentage (+ SE) of explants giving calli with organogenic response in medium NB 0.1/1.0 Cu CM with 100 mg/l kanamycin after coculture with Agrobacterium.

Not precultured (direct)
Precultured
Culture (days)
Control
Agrob.
Control
Agrob.
21
0,0 + 0,0
10, 3 + 2, 3
0, 0 + 0, 0
2, 5 + 2, 3
35
1, 7 + 1, 7
21, 1 + 3, 1
0, 0 + 0, 0
4, 9 + 1, 5

Both kinds of explants experienced a progressive increase of morphogenetic response through the successive subcultures. But this increase was significantly higher in the non precultured explants (21%) than in those precultured for 6 days in the callus-promoter medium (5%) . This difference could be due to the distinct differentiation pattern of the cells belonging to both kinds of tissues, together with the different expression of the genes GUS and NPTII according to the type of promoter which they carry. This fact has already been reported by Jefferson et al., (8) in their work on tobacco when they found significant differences between the different cell types and even among the cell cycle phases in their response to transformation with Agrobacterium. De Kathen and Jacobsen (4) working with Pisum sativum also found different infectivity of the bacterial strain depending on the organ or tissue of the plant.

All the organogenic calli formed either in the first (21 days) or in the second (35 days) subcultures were transferred to the developing medium NB 0.01/0.1 Cu with 100 mg/l kanamycin. Nevertheless, in partial experiments, some of them were transferred to medium without selective agent and no differences were found in the subsequent development of shoots, suggesting that the initial calli, selected for its growth in the presence of kanamycin, came from transformed cells.

All shoots developed in this medium were transferred to the rooting medium MB3 with 100 mg/l kanamycin. Although in previous experiments with tobacco (data not shown) the transgenic plants were easily rooted in this medium, in this case the plants of Cucumis anguria had a very low growth and after 20-30 days had not rooted. Since the NPTII gene is under the nos promoter, it is possible that, according to the above mentioned authors, we had a differential expression of this gene between plant species or even within the different tissues of our species, it being very low or almost nil in its root primordia, making them sensitive to the antibiotic. Therefore, we had to root the plants in medium without kanamycin and we checked the presence of the reporter gene (GUS, which is under the control of the 35S promoter with wider range of expression in the whole plant), in almost all the regenerated plants. The results of this study is summarized in Table 2.

Table 2. Partial and total number of organogenic calli, regenerated plants and GUS-assayed plants coming from the transformation of cotyledon explants (direct or precultured) of Cucumis anguria var longipes.

 
After 21 days
After 35 days
Total
Direct
Precult.
Direct
Precult.
Direct
Precult.
Selected calli
25
4
21
4
46
8
Regenerated plants
107
13
198
62
305
75
GUS-assayed
95
5
153
57
248
62
GUS+ plants
3
0
4
3
7
3

From the 400 (direct + precultured) explants put in coculture with the bacteria, a total of 54 (13.5%) were selected for its growth in the presence of kanamycin. From these calli a total of 380 plants were regenerated in selective medium containing kanamycin and 210 (81.6%) were assayed for the GUS test, giving a positive result with only 10 (3.22%). The origin of these 10 plants corresponds to 7 out of the 54 selected calli (12.9%), so that the final yield was 7 transformed calli out of the 400 explants treated (1.75%). The differences found in the percentages of expression of both markers (particularly the low percent of plants GUS+) could be due to several reasons. Thus, Horsch et al (6) highlighted the need to be careful when transformation is applied to plants other than tobacco or petunia, because of the possible apparition of 'scapes' (shoots that grow in the presence of kanamycin but that do not contain the foreign DNA) due to loss of expression of the gene (or even loss of DNA) during the development of shoots, or to an incomplete selection by cross protection of wild-type cells by transformed cells nearby.

On the other hand, Jansen and Gardner (7), working with petunia, observed great differences between the transient and the stable expression of the markers, the former being 1000 times higher than the latter. It is possible that the extraordinary morphogenetic potential of our species results in the development of transiently transformed green shoot-buds (resistant to kanamycin) which, in fact, are not stable transformants and which, therefore, maybe do not express GUS activity at the level of mature plants. For their part, Colby et al. (2, 3) reported that transformation in Vitis only took place in cells adjacent to the regeneration zones of the calli. For the same reasons it is also possible that, in our case, non-regenerative cells were transformed and, by cross protection, the contiguous non-transformed regenerative cells became plants apparently resistant to the antibiotic.

Lastly, although the position of both markers in the T-DNA is very close, the lack of correlation between the expression of such genes has been suggested by van Wordragen et al. (16) in their work on Chrysanthemum, where they found that the levels of expression of GUS and NPTII varied among calli and not all of those presenting kanamycin resistance exhibited GUS activity.

Conclusions

Agrobacterium tumefaciens strain LBA4404 with the pBl121 plasmid has been shown to be ineffective in cotyledon explants of Cucumis anguria var. longipes. Apparently, the cell type or its state affects such infectivity as shown by the different number of calli selected for its growth in the presence of kanamycin depending of whether the explants have been precultured or not. On the other hand, growth and regeneration capabilities in the presence of kanamycin does not seem to be a suitable index of the transformation efficiency in this species, while the expression of GUS activity in the regenerated plants appear to be as more reliable or advisable index.

At present, experiments aimed at the molecular detection of NPTI gene, either in plants GUS+ or in those not expressing this marker, are in progress. The results will help to clear up if the plants showing an apparent resistance to kanamycin but without GUS activity are true scapes or if a differential expression of both genes (or even a partial loss of DNA fragments) has occurred. Other experiments addressed to increasing the transformation rates in this and other Cucumis species are also being carried out. However at the moment, the plants already obtained in this work are going to be used as genetic markers in experiments of protoplast fusion C. melo (x) anguria.

Additionally, this transformation technology applied directly to the cultivated species should give us a powerful tool for the introduction of known valuable traits (such as virus or herbicide resistance, male sterility, etc.) in this group of important crop species.

Acknowledgements

The authors are grateful to Dr. A. Granell, from the Instituto de Agroquimica y Tecnologia de los Alimentos (CSIC, Valencia, Spain) for the cession of the bacterial strain and for his cooperation and helpful advice in the realization of the work. This work has been carried out with the financial support of the CICYT (COmmission Interministerial de Ciencia y Tecnologia, Project BlO89-0446) from the Spanish Government. R. Santana is grateful for her grant from the Autonomous Government of the Valencian Community (Spain).

Literature Cited

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