Direct genetic transformation of Hibiscus sabdariffa L

Transgenic Hibiscus sabdariffa plants have been produced by a tissue culture independent method using Agrobacterium tumefasciens transformation procedure. Embryo axes of mature seeds with one cotyledon excised were infected by immersion in a suspension of Agrobacterium LBA 4404 strain culture that carries pBal plasmid with β-glucuronidase p35SGUSINT and plant selectable marker Neomycin Phospho-Transferase gene (nptII). Following a 24 h co-cultivation with Agrobacterium strain and decontamination with cefotaxime, embryos were grown on soil rite containing MS medium added with a killer concentration of kanamycin (100 μg/ml) during 4 weeks at room conditions and thereafter transferred to greenhouse. 54.3% of the seedlings grew well on the selective medium; 68% of the explants excised from putative transformed plants were found to be GUS positive. After 60 days evaluation point, the assessment of the transformation by PCR revealed that H. sabdariffa line tested, carried the nptII gene.


INTRODUCTION
Hibiscus sabdariffa is a crop widely cultivated in Sub Saharan Africa, growing on sandy soils after the harvest of the main crop (groundnut or millet) for additional income in rural areas.The crop is now cultivated in periurban areas due to the great demand.The economic importance of the species for African farmers in income generation is more and more emphasized.
The non dehiscent and fleshy fruit containing anthocyanic pigments is largely used as tea and refreshing beverage but also as natural dye.The calices (Bricage, 1985) contain high content of anthocyanic pigments, cyanidin (29%) and delphinidin (70.9%).For this reason the crop has a great economic potential in sub-Saharan Africa (TDC, 1997).
The crop is subject to attacks by Phytophtora parasitica (Follin, 1966), which is the main cause of disease in early  (Sadio, 2000).Callus culture for secondary metabolites production showed that only cyanidin can be produced through young explant callus (Mizukami et al., 1988).Hibiscus improvement programs aimed at the development of varieties resistant to insect and viral diseases or with a high content of anthocyanic pigments.Modification of Hibiscus genome using genetic engineering methods would facilitate rapid development of new varieties with traits that confer resistance to virus and insects or new floral varieties for production of natural dye.An efficient in vitro plant regeneration method is often considered as a prerequisite before transformation.A potential limitation of this tissue culture method is the availability of a regeneration protocol by callus; in addition somaclonal variation events generated through tissue culture may induce unwanted mutation (Venkatachalam et al., 2000).Direct transformation protocol targeting meristems and embryos without an in vitro phase is fast and advantageous by reducing the effort needed to obtain a reasonable number of transformed plants (3 months instead of 9 months).
We report here a rapid and efficient method for producing transgenic plants from H. sabdariffa embryos, which does not require in vitro regeneration.

Plant material
Seeds of H. sabdariffa were soaked in Bavistin (0.1%) for 30 min, rinsed with distilled water, then soaked again in HgCl 2 (0.1%) for 6-7 min and thoroughly rinsed with sterile distilled water.After an imbibition period (8 h), the seed coat were removed, and the two cotyledons kept.The embryonic axes were also used for inoculation with Agrobacterium strain.

Bacterial strains and vectors
Agrobacterium strain LBA 4404 strain containing pBal plasmid with p35SGUSINT and plant selectable marker nptII was used for inoculation.200 to 500 µl of an Agrobacterium culture taken from the stock were suspended in 25 µl of 2YT medium.The Agrobacterium culture medium was added with 50 µg/ml Kanamycin and 25 µg/ml of rifampicin and grown overnight on the shaker (150 rpm) at 28°C until the bacterial culture reach exponential phase of growth.Infection 75-100 embryos were incubated in Agrobacterium culture on rotary shaker (150 rpm) for 6 h, co-cultivated for 24 h in agro-culture then blotted dry on paper filter and cultivated on autoclaved soil rite containing M-S/2 mineral elements (Murashige and Skoog, 1962) with a killer concentration of 50µg/ml kanamycin.After 25 days on selection medium containing antibiotics, green and healthy plants were transferred in pots and let grown in greenhouse with the same selection pressure.Plants were assessed by histochemical GUS test and PCR.

Histochemical Assay
Hibiscus explants inoculated with pBal containing GUS intron were tested.To identify GUS positive tissues from plants growing on selection medium, hypocotyls (1.5 cm) and apex were excised.The tissues were incubated overnight at 37°C in 2 ml of an aliquot from 5 mg Xgluc, 250 µl DMFO, 8.94 ml of 0.1 M phosphate buffer pH 7, 500 µl of potassium ferricyanide, 500 µl of potassium ferrocyanide (Jefferson et al., 1987).After staining, explants were soaked in 70% ethanol for bleaching and stored in methanol.

PCR
Three to four youngest and upper leaves were isolated from one month old putative transformed plant grown in greenhouse and ground in liquid nitrogen in a mortar using a pestle; genomic DNA was extracted according to a modified CTAB procedure (Saghai-Maroof et al., 1984).DNA extracted from fresh tissues was then dissolved in 20 µl of TE buffer containing RNAse.After 30 min, the DNA was completely dissolved and store at -20°C.
For PCR analysis of nptII gene in the transformants, 2 primers were used: 5′-GAG GCT ATT CGG CTA TGA CTG-3′ and 5′-ATC GGG AGG GGC GAT ACC GTA-3′.PCR were performed in a total volume of 25 µl.The PCR products were later analyzed on an  agarose gel (1%) dissolved in 1X TAE buffer.Bands are viewed on UV transilluminator.

Efficiency of the transformation protocol
After 3 days of co cultivation in darkness, mature Hibiscus embryos isolated from germinated seedlings cocultivated with Agrobacterium started to germinate but on selection medium, they lacked chlorophyll, staying etiolated for one week.The transformed plants started turning green after 10 days on selection medium and developed thereafter in green healthy plants.The cultivated embryos produced plants in 25 days culture on selection medium containing 50 µg/ml of kanamycin.Only putative transformants grew well on the selective medium containing lethal dose of kanamycin.Non-transformed embryos did not germinate, becoming bleached rapidly and dying.The rate of putative transformation in Hibiscus was 60.46%.

Expression of GUSINT
Immunological essay was performed for Hibiscus inoculated with pBal containing GUS intron.The analysis of the presence and expression of uidA gene was done 10 days after co-cultivation on different parts of the seedlings (hypocotyls, cotyledons and apex).Hypocotyls tissues showed a dark blue color and were GUS positive.Histochemical activity was also localized both in apex and cotyledonary meristems.On 25 putative transformed plants analyzed, 17 were found GUS positive.

Detection of nptII gene in plant genomic DNA by PCR
NptII expression in transformed Hibiscus lines were tested by PCR.Analysis carried on 5 Hibiscus randomly selected plants, showed amplification of nptII gene on 3 lines (Figure 1).The plants grew very well and set flowers and fruits.

DISCUSSION
The development of a fast procedure of direct (or in planta) plant transformation is the main result achieved in these experiments.We have shown that this procedure is efficient in H. sabdariffa tested with interesting rates of transformation.In Arachis hypogea, Rohini and Rao (2000) reported a direct transformation after wounding with 5 lines transformed on 150 tested.Other reports dealing mainly on Arabidopsis (Bechtold and Pelletier, 1998), Medicago and soybean in planta transformation using vacuum infiltration and flower dipping pointed out the low rate of transformation and the variability of the results (Birch, 1997).
In H. sabdariffa this is the first report on genetic transformation in planta, with several of the plants tested expressing the transgene.The rate of transformation may suggest that there is a strong interaction between the plant genotype and the strain (Hooykaas and Schilperoot, 1992).
This study has shown for the first time that a recombinant gene can be integrated in H. sabdariffa following A. tumefasciens mediated transformation.Further optimization of the protocol is necessary to obtain a high yield of transgenic lines in the progeny.This strategy of direct transformation through embryonic axes, established in peanut (Rohini and Rao, 2000), is simple fast and can contribute significantly to genetic improvement of Hibiscus cultivars in sub-Saharan Africa.

Figure 1 .
Figure 1.H. sabdariffa PCR showing 3 lines with nptII positive bands out of 5 lines tested.