**Acknowledgements**

*Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro*

requiring further studies.

formation.

**5. Conclusions**

High concentration of sorbitol or mannitol will enlarge the distribution of starch granules. It may link to the increase of the shoot organogenesis area [5]. However, the physiological functions and mechanisms of starch accumulation still remained,

Moreover, the accumulation and metabolism of soluble sugars and starch can be induced by AnA treatment (anthranilic acid and ABA supplemented into the medium together) to replace osmotic stress treatment. High levels of endogenous IAA and ABA at the same time are necessary during HRC induction. Both of them need to decreased suddenly in few days are also an important criteria for further shoot regeneration [5]. To link these metabolic changes with phytohormone regulations, we also introduced auxin transport inhibitor TIBA during callus induction and found that it will inhibit carbohydrate accumulation and result in low shoot regeneration frequency. However, ABA signals seemed to be promoted under TIBA treatment [8, 11]. It is still not clear whether exceeding ABA signals will turn into negative regulator on HRC induction, but these results still indicate that there must be interactions among auxin, ABA, and carbohydrate metabolisms on HRC

Inducing regenerate tissues from pluripotent cells is a fascinating event. So far, botanists have already shown that they were able to get regeneration plants from callus in many plant species [49, 65–67]. However, why and how plants achieve this process is still unknown, especially in molecular levels. Here, we propose a hypothesis among phytohormone, osmotic stress, and carbohydrate metabolisms on HRC induction based on current knowledge and our findings (**Figure 2**). According to our model, levels of endogenous IAA upregulated by osmotic stress treatment can promote sugar uptake via CIN and SUT, which result in carbohydrate accumulation during callus induction stages. Similar to auxin, endogenous ABA level is also enhanced under osmotic stress, thus modulating starch accumulation during formation of HRC by downregulating α-amylase activity. Our studies indicated that exogenous auxin or ABA treatment alone is not sufficient for embryonic or organogenic callus formation, which only increased the plant regeneration rate for 35 and 5%, separately [5]. However, when we combine both ABA and anthranilic acid treatment together, the regeneration frequency can be promoted to 80% similar to osmotic stress treatment, suggesting that there must be some interaction between these two phytohormones. The roles of accumulated carbohydrates in HRC could be used as osmotic agents for further metabolism changes or be consumed as an energy

*Working hypothesis of highly regenerable callus induction under osmotic stress treatment in rice.*

**40**

**Figure 2.**

The authors would like to thank Professor Li-Fei Liu, Department of Agronomy, National Taiwan University, to establish the high efficiency of the shoot regeneration system by osmotic stress treatment in rice callus culture. We would like to thank the Council of Agriculture, Taiwan, ROC, for providing the funding to finish this study.
