Optimization of Regeneration System of Tissue Culture and Transformation of 1 Dx 5 Gene without Markers in Wheat

To improve bread making quality of flour and produce transgenic plants free of selectable markers, Immature embryo scutella from Xindong No. 26 were used as explants for establishing an efficient and stable wheat regeneration system, then he linear expressing box of 1Dx5 without selectable markers was transformed into the immature embryo of Xin Dong No. 26 via particle bombardment. The result showed that MS medium containing 2, 4-D 1.5 mg/L and Dicamba 0.5 mg/L for callus induction, R medium containing ZT 1 mg/L and 2,4-D 0.01 mg/L for callus differentiation were the best efficiency, the differentiation frequency of callus was 90%; Transformed plants were screened by PCR, three transgenic plants were detected among 1000 transformed plants, only yielding the transformation rate of 0.3%. The compositions of HWM-GS were analyzed by SDS-PAGE. The HMW-GS gene 1Dx5 was expressed in some seeds of transgenic plants. Our studies lay the foundations for obtaining marker-free transformants using the linear gene via particle bombardment.


INTRODUCTION
Through the biological technology and genetic engineering method to improve wheat agronomic characters had been widely attentioned (Lazzeri et al., 1997) and become conventional breeding auxiliary mean (Birch, 1997).Using this technology, the researchers could transfer the valuable genes into the local varieties of wheat so that produced transgenic wheat varieties which had production value (Patnaik and Khurana, 2001).It was well known that land salinization and lower wheat processing quality always restricted the Xinjiang's grain production.In order to make the rapid development of wheat industry in arid areas, it was urgent to breed new traits of salt-tolerance wheat which improved processing quality.Research showed that HMW-GS had molecular weight for 90~147 kd andcontained disulfide bond, which played a decisive role in bread elasticity (Ng and Bushuk, 1988) and baking quality (Shewry et al., 1989).1Ax1, 1Dx5, 1Dy10 were considered the high quality subunit genes (Shewry et al., 1992).Therefore, the wheat which had these subunit genes and can be expressed had a broad application prospects in the production.In order to use the high quality 1Dx5 gene of HMW-GS in wheat, the author had separated, purified and cloned the subunit gene from high quality variety of Xinjiang (Xiao-Yu, 2011) and transformed it into common wheat planted widely to improve their processing quality.At present, mediated genetic transformation with Micro projectile bombardment had been matured (He et al., 1998).But it was need to establish an effective tissue culture system (Fennell et al., 1996).Along with the establishment of plant tissue culture technology, wheat genetic transformation research had made a great progress.But significant differences between the different genotypes of wheat and the low regeneration frequency still seriously restricted the wheat genetic engineering research development.
So, in this study, immature embryo scutella of Xindong No. 26 was taken as research materials for establishing an efficient regeneration system.With the methods of particle bombardment to transform the 1Dx5 gene into salt-tolerant variety would lay a foundation for wheat processing quality improvement.

MATERIALS AND METHODS
The study was conducted in Research Room of Molecular Biology and Informatics, Xinjiang Normal University from 2009 to 2012.The wheat receptor material was salt-tolerant wheat of Xindong No. 26 in xinjiang and positive control was L88-6.They were all sowed in Xinjiang Normal University experimental field.
Plasmid pMon 530 and E coli strain DH5α were saved in laboratory.
In induced phase, immature embryo scutella callus were cultured in dark and temperature was maintained at (25±1) °C.When cultures were shifted to differentiation phase, temperature was maintained at (25±1) ºC with 16 h light (about 2000 lux) cycle in every 24 h.Methods: Took ears of wheat of flowering 15 days after pollination.Picked off the immature seed andsterilized by washing with 1% HgCl 2 for 12 min followed by 3-5 washes with sterilized distilled water.Took the immature embryo carefully, picked off the plantule, turned up the scutella and inoculated it on the induction medium.After 30 days, these induced callus were inoculated to the differentiation mediums according to the groups.Counted callus induction rate after 30 days on the induction medium and counted differentiation rate in 30 days after transferred to the differentiation medium.The specific statistical analysis method referred to Liu (2005).In addition, average rate of emergence (%) = The number of seedling differentiation/The number of inoculation of explants×100%.
Ten to fourteen days after plant flowering, stripped out of the seeds, disinfected the surface, got rid of the immature embryo and took the scutella as the test material.Osmotic treatment of immature embryo scutella was done for 4 h with 0.4 M mannitol after it two days preculture.Bombardments were carried out with particle bombardment (PDS 1000/He, Bio-Rad) under a vacuum of 27 inches of Hg and the conversion process refers to improvement methods of Rasco et al. (1999).The variables included rupture disc pressures (1,100 psi), distance from rupture disk to macrocarrier (6 cm), type of micro carrier (gold), size of micro carrier (1.0 μm diameter of gold particles), number of bombardments per Petri plate (1), precipitating agent of ackage the bullets (0.1 M Spermidine and 2.5 M CaCl 2 ).After bombardment, scutella were kept on medium supplemented with 0.4 M mannitol for 16 h then transferred to induction medium without mannitol.The Experiment designs two controls: gold bombardment (without exogenous gene) and no bombardment.
The PCR reaction conducted as followed program: An initial denaturation at 9°C for 5 min, followed by 35 cycles of 94°C for 30s, 6 for 30s and 72°C for 90s and a final extension step at 72ºC for 7 min.The a °C amplified products were separated on 1% agarose gel and photographed using Gel Documentation System.
Harvested seed of positive plant separately, chipped half off spring seeds without germ and picked with study to crush by sample clamp.As mentioned, to extract its high molecular weight glutenin subunit for SDS-PAGE analysis (Zhu et al., 2008).

RESULTS AND DISCUSSION
Immature embryo scutella were cultured on ten kinds of callus induction medium (had different concentration of 2, 4-D and Dicamba).Then, transferred the callus to three kinds of differentiation medium with 2, 4-D 0.01 M and gradient concentration of ZT.
Furthermore, seedling which differentiate in 1M ZT would growth better than others and is more feasible to transplant.
This experiment results also showed that combination of 2, 4-D and Dicamba had profound effect on differentiation frequency of callus tissue.Different combination ratios of 2, 4-D and Dicamba had significant effects.On the combination of 0.5 M 2, 4-D and 1.5 M dicamba, callus induction and green plant regeneration rate were higher than any other combination.The green plantlet frequency and the average rate of seedling emergence were 79.33 and 100%, respectively (Table 2).The result showed that the combination of 0.5 M 2, 4-D and 1.5 M Dicamba was more advantageous to the differentiation of induced callus.
Base on the best induction medium (0.5 M 2, 4-D and 1.5 M Dicamba), R medium added 1 M ZT and 0.01 M 2, 4-D, the best differentiation rate was gotten.The green plant regeneration rate was 90% (Fig. 1).The regeneration plant was transplanted after the seedling formation.The survival rate was above 95% and these plants can be matured to seed.
Immature embryo was the earliest explants used in the wheat genetic transformation.Many studies suggested that, the induction and plant regeneration ability of immature embryo callus was stronger than the young spike and mature embryo (Lü et al., 2007).Based on immature embryo scutella for transformation receptor, not only the false positive bringed by embryo could be reduced, but also its induced callus had the best plant regeneration frequency compared to other explants materials (Qin and He, 2001).For all the gramineous plants, the regeneration ability of immature embryo scutella was better.Immature embryo scutella were transformed by particle bombardment had achieved success (Birch, 1997).
It was generally acknowledged that 2, 4-D was an essential plant hormone in callus induction (Zhou et al., 2000).Two, 4-D to callus relied on the genotype of wheat (Umer et al., 2009).Some studies found that the induction effect of this research result indicated that the callus tissue differentiation rate was influenced obviously by2, 4-D combined with Dicamba than each of them in solo.The result was the approximate homology with the result of research to Bryl genotype by Przetakiewicz et al. (2003).The reason may be that the combination of Dicamba and 2, 4-D is beneficial to the formation of embryonic callus.Nbaors et al. (1983) was also reported that the 2, 4-D or 2, 45-T used separately would promote the formation of nonembryonic callus.Embryonic callus had regeneration characteristics and could be regenerated plantlet by embryogenesis or organogenesis.Non-embryonic callus only could be proliferation, but not regeneration.
Based on the regeneration system, linear expression box of HMW gene 1Dx5 without selection To determine if the transgene was expressed in transformed plants, the total proteins of seeds of transgenic plants progeny were analyzed by SDS-PAGE.It was showed that 1Dx5 would be expressed in seeds of transgenic offspring to some extent (Fig. 3).This research transformed the linear expression frame without selection markers into wheat immature embryo scutella by particle bombardment.Results showed that 1Dx5 had expressed in several grains of transformed plants.It confirmed previous reports (Uzé et al., 1999) that of the minimum expression box of purpose gene only contained promoter, coding sequence and terminator can be used in wheat particle bombardment operation successfully.Although this transformation system had lower transforming rate compared with Rooke et al. (1999) (1.4%) and Gadaleta et al. (2008) (1.5%), it can improve the safety of transgenic plants.However, UZE co-transformed the wheat gene by the linear segment GUS and bar and the results show that transforming rate of linear fragment was higher than complete plasmid.The different results may be that there no use resistance selection pressure in particle bombardment transforming process.Mullins et al. (1990) etc., also thought that transformed cell would be at a disadvantage competition state with no-transformed cell without selection pressure during genetic transformation.So this transformation system should be optimized to improve transformation frequency.

CONCLUSION
This research established a high efficiency and stability wheat plant regeneration system with the immature embryo scutella as the explants.On that basis, non-selection markers linear 1Dx5 genes was transformed into the immature embryo callus of wheat of Xindong No. 26 in xinjiang and transgenic plants were obtained which 1Dx5 expressed in the offspring seeds.All of that laid a foundation for obtaining the safe new transgenic variety of wheat, which also had salt-tolerant characteristic and high processing quality.

Fig. 1 :
Fig. 1: Plant regenerated on the differentiation medium for 30 days