IN PLANTA TRANSFORMATION OF BUCKWHEAT ( FAGOPYRUM ESCULENTUM MOENCH . )

In order to develop a reliable and rapid transgenic system for functional study of specific buckwheat gene constructs, two different in planta transformation methods were analyzed: vacuum infiltration and infiltration by syringe. The results indicated that the vacuum infiltration method was much more efficient and can therefore be considered the method of choice for buckwheat transformation.


INTRODUCTION
Agrobacterium-mediated in vitro transformation has been routinely used for the production of transgenic plants (Horschet al., 1985), although it is usually time-consuming.Therefore, it is important to develop in planta transformation methods that do not require sterile conditions of tissue culture and enable rapid gene functional analysis.

Common buckwheat ( Fagopyrum esculentum
Moench) is a crop grown mainly in the northern hemisphere.The large amount of lysine and well-balanced content of other essential amino acids characteristic of buckwheat seed storage proteins make this plant highly recommendable for human consumption.Buckwheat is also unique among crops due to the presence of rutin, a flavonol glucoside serving as part of the defense system against oxidative stress.Aside from these traits, there is rising interest in this plant as an object of molecular biology and biotechnology research.Gene expression and functional promoter analysis of specific buckwheat genes could be an element in the process of understanding common mechanisms of gene regulation in plant cells.
Until now buckwheat has been transformed only by an Agrobacterium-mediated in vitro method (M i lj u š -D j u k i ć et al., 1992).This is the first paper reporting a rapid and effective method for in planta transformation of buckwheat.Two different in planta transformation methods were optimized: vacuum infiltration and infiltra-tion by syringe.

Plant material and vectors used
Buckwheat plants (Fagopyrum esculentum Moench) grown in a greenhouse were used for transformation experiments.The two vectors used for transformation -pCAMBIA2301 (Cambia, Australia) and pCAMBIA-PL -were electroporated into Agrobacteriumtumefaciens strain EHA105.pCAMBIA2301 is a strong CaMV35S promoter upstream of the GUS reporter gene, while pCAMBIA-PL is a promoterless GUS reporter gene.

Vacuum infiltration
Vacuum infiltration was performed according to Bechtoldet al. (1993).An overnight culture of A. tumefaciens (10 ml) was inoculated in 1 L of LBmedium supplemented with 150 µM acetosyringone (AS), 50 μg/ml kanamycin, and 10 μg/ml rifampicin.Bacterial cultures were grown overnight at 28°C to OD 600 ranging from 0.8 to 2.1, centrifuged (5000 rpm/RT/15 min), and resuspended in infiltration medium [½ strength Murashige and Skoog macro and micro salts and vitamins, 5% sucrose, 2.6 µM MES, 44 nM 6-benzylaminopurine (BAP), 150 µM AS, 0.02% Silwett L-77, pH adjusted to 5.7].Plants were removed from pots and immersed in 1 L of infiltration medium an hour before vacuum infiltration.Two different vacuum conditions were used for infiltration: pressure of 10 4 Pa was applied for 5 min and 10 2 Pa for 20 min, then released rapidly to increase the infiltration efficiency.After removal from the dessicator, plants were laid on their side for 15 min and wrapped in transparent plastic to maintain humidity.The next day they were uncovered and set upright in pots, but not watered for three days.

Infiltration by syringe
Infiltration by syringe was performed according to Y a n g et al. (2000).Briefly, Agrobacterium cultures were grown overnight at 28°C/150 rpm to OD 600 ranging from 0.8 to 2.1.Cells were collected by centrifugation (5000 rpm/RT/15min) and resuspended in infiltration medium [10 mM MgSO 4, 10 mM MES (pH 5.5), 150 µM AS].The bacterial suspension was infiltrated by applying pressure against the lower side of a young leaf with a needle-less syringe.After infiltration, plants were kept covered with transparent plastic bags for two days without watering.

Quantitative GUS assay
The level of transient GUS activity monitored by fluorescence GUS assay (J e f f e r s o n, 1987) was used as a measure of transformation efficiency.The protein concentration of sample extracts was determined using a BioRad Protein Assay Kit with BSA as the standard.GUS activity was expressed as pmol 4-MU per hour per mg of protein, where 4-MU was quantified with a Versa-Fluor fluorimeter (BioRad).

Statistical analyses
The results were statistically analyzed using the SPSS statistical program.Since the obtained values did not show normal distribution, the nonparametric Mann-Whitney test and median values were used for comparison of different groups of samples.

RESULTS AND DISCUSSION
According to numerous papers reporting successful in planta transformation of different plant species, the efficiency of transformation depended on several conditions, namely Agrobacterium culture density, vacuum conditions, and leaf maturity.It was shown that optimal values of these factors have to be determined for each plant species.
For optimization of the vacuum infiltration method for buckwheat, a total of 230 samples were analyzed.The influence of A. tumefaciens culture cell density, as well as pressure conditions and time elapsing between the transformation event and GUS measurement, were examined as factors that could be important for transient transformation efficiency.
Various plant species were successfully transformed using bacterial culture OD 600 values varying from 0.8 to 2 ANAM.BRATIĆ ETAL.136 Fig. 1.Optimization of the vacuum infiltration method.Influence of bacterial culture OD 600 and time elapsing between transformation event and GUS measurement on GUS activity.In all cases 10 4 Pa/20 min vacuum conditions were applied.Data are median values of GUS activities obtained by measuring 10-15 samples for each OD 600 /day.Influence of vacuum conditions on GUS activity.EHA105/pCAMBIA2301 and bacterial culture OD 600 1.1 were used for both examined conditions.GUS activity was measured 6 days after transformation.Clough, 1998).To determine the most suitable bacterial culture OD 600 for buckwheat,we tested transformation efficiency using OD 600 values within the following ranges: [0.8-0.9];[1.1-1.2];[1.3-1.5] and [1.9-2.1],under a pressure of 10 4 Pa/20 min.To find out when maximal GUS activity was reached, quantitative assays were performed 3, 5, 6, and 10 days after transformation for all tested OD 600 values.Figure 1a shows the GUS values obtained for all examined conditions in plants transformed with EHA105/pCAMBIA2301 or with pCAMBIA-PL (negative control).On the third day after transformation, the detected GUS activity was low, but significantly higher than the GUS value measured in plants infiltrated with EHA105/pCAMBIA-PL, except for the case of OD 600 [1.8-2].Maximum GUS activities for all tested OD 600 ranges were reached on the 5 th and 6 th days, but it was significantly decreased on the 10 th day.The most suitable bacterial culture OD 600 was [1.1-1.2]measured 6 days after transformation, since it resulted in distinctly higher GUS activity than under any other applied conditions.
As mentioned above, the efficiency of vacuum transformation also depends on the applied vacuum conditions, which have to be determined for each plant species.For example, tobacco was successfully transformed under conditions of 8x10 3 Pa/20 min (W a n g et al., 2002) and wheat under conditions of 10 5 Pa/60 min (Amoahet al., 2001), while for Arabidopsis in planta transformation different vacuum conditions in a range of from 1.6x10 3 Pa/20 min to 5x10 4 Pa/15 min were confirmed as suitable (B e c h t o l d et al., 1993;Clough et al., 1998).To define optimal vacuum conditions for buckwheat in planta transformation, we tested two different pressures viz., 10 3 Pa and 10 4 Pa, and durations of 5 min to 25 min of vacuum treatment, applying the previously determined optimal OD 600 1.1.We found that 5 min was optimal for the vacuum pressure of 10 3 Pa, while 20 min was the most suitable for the vacuum pressure of 10 4 Pa (data not shown).Comparison of these two vacuum conditions showed that 10 4 Pa/20 min was the most suitable vacuum condition for buckwheat transformation, as the corresponding median value for GUS activity was 14 times higher than for leaves infiltrated under 10 3 Pa/5 min (Fig. 1b).It was also noticed that many leaves infiltrated under 10 3 Pa/5 min wilted, while leaves treated using 10 4 Pa/20 min showed faster recovery.
For optimization of the syringe infiltration method, the influence of bacterial culture OD 600 was examined.GUS activity was measured 5 and 6 days after transformation of buckwheat, bearing in mind that maximal activity was reached within that period for vacuum infiltration.The results are summarized in Fig. 2a.For all examined OD 600 ranges, it was shown that GUS activities were significantly higher than in plants infiltrated with the EHA105/pCAMBIA-PL strain.In contrast to vacuum infiltration, syringe transformation efficiency did not vary much among the different OD 600 ranges higher than 1, as the corresponding values were not significantly different at p<0.05.Two groups of OD 600 ranges were found to ) obtained from 15 samples.Comparison of two different transformation methods under their optimal conditions (vacuum infiltration: OD 600 1.1 / 6 th day; syringe infiltration OD 600 1-2 / 5 th and 6 th day).
have statistically significant differences in measured GUS activity.Buckwheat transformation was more efficient for the group within the OD 600 range of from 1 to 2, since the corresponding median values were approximately twice as high as those obtained for OD 600 0.8 -0.9.
The results obtained under the defined optimal conditions for both described transformation methods are compared in Fig. 2b.Vacuum infiltration appeared to be far more efficient, as the median value obtained for GUS activity was 57.3 times higher than that obtained with infiltration by syringe.

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B e c h t o l d et al., 1993; K a p i l a et al., 1997; Amoahet al., 2000; T r i e u et al., 2000; B e n t and

Fig. 2 .
Fig. 2. Optimization of the syringe infiltration method.Influence of bacterial culture OD 600 on transformation efficiency.Data for each OD 600 /day represent median values of GUS activities (pmol MU mg -1 h - 1