Ovary-drip Transformation Method Of Soybean (Glycine Max L.) And Its Application In The Transformation Of Soybean With Salt-tolerant Gene (AlNHX1)

The rapid development of plant transgenic techniques has a large impact on traditional crop breeding.However,there is a concern that transgenic plants could affect food and environment safety,particularly the problem associated with biosafety posed by the vector, such as its backbone sequence and the resistant marker that are incorporated into the vector. Particle bombardment and Agrobacterium-mediated transformation are two commonly used methods for obtaining transgenic plants.However,these two methods have their limitations in soybean,resulting in relatively inefficient production of transgenic soybean.Alternative transformation method such as the pollen-tube pathway has also been tried in soybean,but without much success in engineering transgenic soybean due to its low transformation frequency and reproducibility.Moreover,soybean(Glycine max L.) is classified as a salt-sensitive glycophyte.As for soybean,salinity stress inhibits seed germination and seedling growth,and reduces nodulation,biomass accumulation and seed yield.Thus,if salt-tolerance gene could be introduced into soybean by the plant gene technology,the salt-tolerance of soybean could be improved,and the effect of its salt-tolerant capacity could be increased.To increase the transformation frequency and reproducibility,we established and optimized a tissue culture-independent plant transformation method called ovary-drip method, in which a gene cassette(35S CaMV promoter,smGFP open reading frame,and NOS terminator) was applied directly to a wound site induced on the ovary of soybean.We observed the growth of the pollen tube in different periods after self-pollination.Using smGFP gene as a model gene we traced the smGFP gene by FITC(fluorescein isothiocyanate)-labeled DNA solution with an optimal composition of 0.05%Silwet L-77 and 8%sucrose.The transformation frequency as determined by PCR reached 3.18%during 6～8 h period after self-pollination.Southern blot analysis of the primary transformants showed that the integration of smGFP gene.The transgenic plants exhibited a high-level of smGFP expression,which was visible in the embryos of the transgenic soybean.PCR and Southern blot analyses of the T₁ generation of these transgenic soybeans showed that the smGFP gene was inherited by the progenies. To validate the reproducibility and genetic stability of the ovary-drip transformation,a marker- and vector-free GUS gene cassette was introduced into soybean.The results from histochemical analysis and PCR screening of To generation showed that 12 out of 340 detected immature embryos were GUS positive,and six out of 180 transformed plants were PCR positive and GUS positive.This corresponds to a transformation frequency of 3.53%for the transformed embryo,and 3.33%for the transformed plants.Southern blot analysis confirmed that GUS gene was integrated into the soybean genome.The GUS staining,PCR and Northern blot analyses indicated that GUS gene was inherited by the progenies.Among six lines of T₁ generation,only one showed a Mendelian segregation for the GUS inherited GUS gene.Having confirmed the reproducibility of the ovary-drip transformation method in soybean,the enhancement in salt tolerance of soybean was investigated by transforming soybean with a Na⁺/H⁺ antiporter gene(AlNHX1) cloned from Aeluropus littoralis var. sinensis De beaux.Six out of 200 transformed plants were positive as determined by PCR, corresponding to a positive rate of 3.0%.Three of the positive plants were further examined by Southern blot and RT-PCR.The results indicated that AlNHX1 gene was integrated into the soybean genome and was expressed.The inheritance of AlNHX1 gene was confirmed by PCR and Southern blot and the progenies derived from T₁ generation were examined with respect to physiological and biochemical traits.The results showed that all transgenlc progenies have enhanced salt tolerance.Under salt stress,increases in the levels of Na⁺(29%) and K⁺(～2 fold) were observed in the roots of these transgenic plants compared to the roots of wild-type plants. Decrease in Na⁺ level(21%) and K⁺ level(～2 fold) were also observed in the leaves of the transgenic plants.A higher ratio of K⁺ to Na⁺ was maintained in the leaves of the transgenic plants.In the leaves of the transgenic plants the osmotic potential decreased by 39%,while relative water increased by 9%,chlorophyll contents by 25%,and photosynthesis ability by 94%.Transgenic plants also showed an increase in SOD(45%) and POD(69%) activities over wild-type plants.The results showed that the AlNHX1 gene was functionally expressed in soybean,with the effect of increasing its salt-tolerant capacity under salt stress condition.In this study,the ovary-drip method with higher transformation frequency and reproducibility was established for soybean.In addition,a marker-free and vector-free AlNHX1 gene cassette was transferred into soybean via ovary-drip method.The gene was functionally expressed in soybean with the effect of increasing its salt-tolerant capacity under salt stress condition.The ovary-drip method provides a transformation method for introducing functional gene of interest into soybean,and possibly for other plants as well.