Wild ornamental germplasm exploration and domestication based on biotechnological approaches. In vitro colchicine treatment to obtain a new cultivar of Scoparia montevidiensis
Alejandro S. Escandón*
Financial support: The present work was supported by the INTA JICA agreement, project: Argentina Floriculture Development.
Keywords: chimera, flow cytometry, micropropagation, ornamental plants, tetraploid.
The genus Scoparia
is native from
genus Scoparia belongs to family of the Scrophulariacea.
So far, 10 native species from
This genus shows an interesting diversity in shapes and colors of the flowers, as well as different growth habits. For instance, S. montevidiensis var. montevidiensis, S. montevidiensis var. glandulifera, S. nudicaulis, S. hasleriana are herbaceous plants, whereas S. dulcis is a sub shrubby plant, and both varieties present a profuse growth with abundant yellow blossom. S. dulcis has white flowers, and the flowers of S. hasleriana and S. nudicaulis are sky blue. However, flower size represents a problem in terms of their potential commercial value because in all of five Scoparia species they are too small. The development of polyploids can be a useful and valuable tool to improve this trait in breeding programs (Notzuka et al. 2000).
Polyploidization was used successfully to increase the size of flowers, intensify the colours of leaves and flowers, modify plant shape and restore fertility in ornamental species (Horn, 2002).
Tissue culture using colchicine treatment is an interesting biotechnological alternative tool for the domestication and early exploration of wild germplasm with ornamental potential. In the present study we describe the in vitro micropropagation protocol for four species (five different accessions) of Scoparia spp, as well as the development of a new variety of S. montevidiensis using in vitro colchicine polyploidization as a breeding tool.
Nodal segments of S. montevidiensis var. glandulifera were first sonicated during 20 min and then disinfected by immersion in 70% ethanol for 1 min and then in a solution containing 25% sodium hypochlorite: 0.01% Tween 80 during 25 min. Finally they were rinsed three times with distilled and sterile water.
explants were cultured onto a hormone free MS medium (Murashige
and Skoog, 1962) and served as a source for the explant used to
study the behaviour of S. montevidiensis var. glandulifera
under different hormones combinations. The medium used for the culture
of the nodal segments was MS supplemented with 20 g/L sucrose, 7 g/L
agar, and the following growth regulator concentrations: 0.0; 0.25;
0.5 and 1.00 mg/L NAA and BAP, in all possible combination. The pH
was adjusted to 5.7 with KOH. The photoperiod was 16:8 under an irradiance
of 3,000 lux. The recovered shoots were transferred monthly to the
same fresh medium except for the rooting step. Three or four centimetres
length plantlets were transferred to the rooting medium (MS hormone
free). The rooted plants were acclimatized according to Escandón
et al. (2003), that is, the rooted plants were transferred to
Nodal segments from in vitro plant of S. montevidiensis var. montevidiensis were submerged in 1% DMSO solution containing the following doses of colchicine (v/v): 0.0; 0.1; 0.05; 0.01 and 0.001% (24 and 48 hrs). As control treatments, a group of nodal segments was untreated, and other segment groups were submerged in water or in 1% DMSO (water solution). The number of explants per treatment was 15. The culture medium was MS supplemented with 0.25 mg/L BAP. The culture conditions and the acclimatizing protocol were those mentioned in the above section.
ploidy level was determined using the flow cytometer (Partec, CA),
following the commercial indications, that is, approximately 0.5 cm2
of leaf tissue were chopped with a sharp razor blade submerged in
0.5 ml nucleus extraction buffer (HR A solution, Partec, CA) and then
incubated in the same buffer during 1.5 min. After filtered, the solution
was incubated 1 min with HR B,
Once established under greenhouse conditions, the recovered treated plants were phenotypically analyzed. The diameter of flowers and stems and the size of the leaves were gauged. For the flower diameter 12 well developed flowers were measured. The stem diameter was measured at the height of the third leaves pair. Also, the third leaves pair was chosen to measure the ratio length/ wide to establish the size of the leaves.
Statistical analysis was performed using ANOVA and Tukey test (95%) supported by the software Statistica 2.0.
Table 1 shows the results obtained with the nodal segments of S. montevidiensis var. glandulifera. In the treatments containing NAA (alone or combined with BAP), the callus induction was the main response of the culture. The treatments containing BAP alone and the hormone free treatment showed shoot development. Significant differences in the shoots multiplication rate were detected between the treatment containing 0.25 mg/L BAP (9.04 shoots/explant) and the other treatments. The treatment containing 1.0 mg/L BAP showed callus production and a very important tissue vitrification. In the treatments containing 0.5 and 0.25 mg/L BAP shoots and callus development were also detected. Although the callus production was important in the treatment with 0.5 mg/L BAP, no vitrification was detected in this treatment. Either callus formation and/or vitrification process were not found in the hormone free treatment. All the recovered shoots rooted in hormone free MS medium. The progress of the in vitro culture of S. montevidiensis var. glandulifera onto a MS medium supplemented with 0.25 mg/L BAP is showed in Figure 1. Figure 1a shows the original explant, a disinfected nodal segment with two nodes at the early times of the culture. A well developed nodal segment showing several shoot and a very little callus development is showed in the Figure 1b. Figure 1c, shows a rooted shoot at the end of the in vitro culture, and the acclimated and flowering plants growth under greenhouse conditions is showed in Figure 1d.
The responses of the five Scoparia accessions under the culture conditions proposed in the present work are showed in Table 2. Under the culture conditions tested, no significant differences were found between S. montevidiensis var. glandulifera, S. montevidiensis var. montevidiensis, S. dulcis and S. nudicaulis. In fact, the mean of shoots per explant for these species oscillated between 10.2 for S. nudicaulis and 12.0 for S. dulcis. The exception was S. hasleriana that showed little callus proliferation, followed by browning of the callus and ending with explants death under the assayed culture conditions.
and viable plants of S. montevidiensis var. montevidiensis
were recovered after the colchicine treatment. Table
3 shows the means of shoot per explant obtained from the different
colchicine treatments. Except for the treatments 0.1% colchicine,
24 and 48 hrs, the others treatments oscillated from 9.0 shoots/explants
(0.05% colchicine/48 hrs) to 12.12 (water control) and no significance
differences were found between them. The unique treatments showing
significant differences in relationship with the controls were those
with the 0.1% 24 and 48 hrs colchicine doses, the means of these treatments
were 6.85 and 6.57 respectively. The number of flow citometry analyzed
plant, the number of chimeric (2X/4X) plants detected and the solid
tetraploid (4X) plants obtained by colchicine treatment with S.
montevidiensis var. montevidiensis are also showed in Table
3. From a total of 379 analyzed plants, 15 chimeric plants were
detected among all colchicine treatments except from the dose 0.1%/48
hrs. A total of four solid tetraploid plants were detected, two with
the 0.001%/24 hrs colchicine dose, one with the 0.001%/48 hrs dose
and the last one with 0.05%/24 hrs dose. Figure
2 shows an example of peak reading obtained by flow citometry
analysis, the right
Figure 3a, b, c compares the appearance of the tetraploid and diploid plants. It is possible to observe differences in the foliage (Figure 3a), being more abundant those of the tetraploid plant (arrowed). The Figure 3b shows the different sizes of a tetraploid flower (arrowed) and a diploid one. In the upper lane of Figure 3c are tetraploid leaves that showed to be larger than the diploid ones (Figure 3c, lower lane). The Figure 3d shows a flowering tetraploid plant with an interesting relationship flower/foliage.
The differences measured between the organs of tetraploids and diploid plants are showed in Table 4.
All the analyzed traits (diameter of stem and flower and leaf size) showed clearly that the tetraploid plant named as S1 was significantly larger than both, the tetraploid S3 and the control plant, being the means of the leaf size (234.27 mm2 for S1, 99.19 mm2 for S3 and 22.4 mm2 for the control one) the variable that showed more dramatic differences among the analyzed plants.
Chromosome duplication or polyploidy, a common phenomenon in ornamental species (Horn, 2002), is associated with an enlargement of organs (flowers and leaves), an intensification of colours, hardier and more robust plants, thicker and more rigid foliage, an apparent increase in the tolerance to different stresses, and the resistance to diseases and pests (Petit and Callaway, 2000). For this reason poliploidization is recognized as a source of evolution and domestication of flowering plants (Van Tuyl and Lim, 2003).
Chromosome duplication is caused by abnormalities during mitosis and it may occur spontaneously in most plants. Although, these disruptions during cell division can be artificially induced with colchicine and thus, in vitro polyploidization was proposed back in the 1960´s as an alternative tool to obtain polyploid plants (Murashige and Nakano, 1966). This methodology was extensively used during the last 30 years in many species such as banana (Baziran and Ariffin, 2002), grapes (Notsuka et al. 2000), blueberry (Lyrene and Perry, 1982), potato (Hermsen et al. 1981), and sugarcane (Heinz and Mee, 1970). Under in vitro controlled conditions polyploidization was applied in several ornamental crops, such as Alocasia (Thao et al. 2003), Rhododendron (Väinölä, 2000; Eeckhaut et al. 2002), Cyclamen (Ishizaka and Uematsu, 1994; Takamura and Miyajima, 1996).
To start with the in vitro polyploidization experiments, the appointment of in vitro micropropagation is required as the first step. The tissue culture experiment in this study showed that Scoparia monteviediensis presents strict hormonal and nutritional requirements. Although the addition of NAA induced callus proliferation in all treatments regardless of BAP concentration, the latter had an apparent impact on the calli aspect. In BAP concentrations up to 0.25 mg/L, calli were compact whereas with concentrations of 0.25 mg/L BAP and in BAP free media, calli were friable. In the hormone free treatments and those with BAP alone, shoots production was the main response of the explants. In this case, multiplication rate varied with BAP concentration, having obtained the best response with 0.25 mg/L BAP (9 shoots/explant). An increment of the citocinine, not only did not improved the multiplication rate, but also induced callus proliferation. At BAP concentrations of 1.0 mg/L BAP, a very important tissue vitrification was observed.
Scoparia spp. rooted easily using the proposed protocol, even with the previous BAP treatment. This, together with the fact that no problems were found either in the acclimatization step, or in the multiplication rate, indicates that this material is suitable for commercial multiplication.
When the protocol proposed for S. motevidiensis was tested with the other Scoparia species, only S. hasleriana did not present a good response. In the same way, satisfactory results were obtained using the same conditions applied here with other Scrophulareaceae genus (data not shown). These results represent an important start point for the evaluation of other Scrophulariaceae species for in vitro shoot multiplication that will contemplate a careful adjustment of BAP concentration to avoid undesirable results such as callus proliferation and/or vitrification process, with no addition of NAA.
There are different alternative techniques to get in vitro polyploid plants. For Rhododendron simsii, Eeckhaut et al. (2002) reported a treatment using a drop of colchicine solution placed between the cotyledons daily during either 3 or 7 days in a 3 weeks seedling obtained in vitro. Takamura and Miyajima (1996) inoculated the tuber sections of Cyclamen persicum by submersion in colchicine solution without shaking for 1, 2, 4 and 7 days. Väinölä (2000) tested successfully an in vitro polyploidization protocol of microshoots of Rhododendron hybrids with synchronized growth by submersion and shaking in different concentration of colchicine solutions.
In the present study the tissue culture experiments showed that Scoparia genus has a good response under the in vitro conditions applied, generating several shoots from one nodal segment, even in the hormone free medium (2.25 shoots/explant). Consequently, when using explants from in vitro plantlets with actively growing meristems the recovering of polyploid individuals is expected.
All the recovered plants (diploid, chimaeric and tetraploid plants) showed the same behaviour during the different stages of the in vitro multiplication protocol.
Under the conditions tested, the colchicine treatment of 0.05%/48 hrs seems to be the maximum dose that did not affect the regeneration capacity of the S. montevidiensis var. montevidiensis nodal segments. In fact, no significant differences were found between the average of shoots per explant of the control and the mentioned treatment. But when the explants were exposed to a colchicine solution of 0.1% during 24 and 48 hrs, the multiplication rate diminished significantly from 12.12 shoots per explant for the control 2, to a value of 6.57 shoots per explant for the treatment 0.1/48. In the same way, in all treatments chimaeric and/or solid tetraploid individuals were recovered, the exceptions were doses where all the recovered shoots were diploid. It is possible that at this colchicine concentrations the damage induced by the alkaloid was turning the affected shoots non viable.
The variation coefficients obtained in the analysis of the ploidy of the peak readings of the Figure 2 shows clearly the difference of the DNA amount between the tetraploid plant and their control.
diameter of stem and flower and leaf size showed in Table
4 are in agreement with descriptions in Figure
The present study is the first report of the application of this biotechnological methodology in Scoparia genus. Tissue culture combined with the polyploidization treatment showed to be a very interesting alternative to obtain the needed variability in Scoparia genus to start a breeding program.
We thank to Carlos Greco and Alberto Acevedo for their critical revision of the manuscript and their suggestions. Also we thank to Sara Ostertag and Martin Saragoiti for their technical support.
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