Study on micro-grafting in vitro of pomegranate

Abstract: 【Objective】Pomegranate (Punica granatum L.) originates from central Asia, is an economically important deciduous fruit crop, cultivates for its delicious edible fruits. In addition, pomegranate fruit has higher levels of antioxidants than the other fruit crops, such as orange (Citrus sinensis) , apple (Malus domestica) , grape (Vitis vinifera) and kiwifruit (Actinidia chinensis) . Antioxidants are potentially beneficial in preventing cardiovascular disease, diabetes, and prostate cancer.‘Tunisa soft-seed'was introduced to China from Tunisa was popular with customers. While low temperature is a limited factor as the‘Tunisa soft-seed'cultivation in northern China. Cold which impaired plant growth and development and reduced productivity is one of the most devastating abiotic stresses in‘Tunisa soft-seed'.Therefore, enhancement of cold tolerance becomes a major subject of considerable research interest over a long period. Some manual measures could be used to enhance cold tolerance, while creating coldresistant cultivars can be an optimal choice. The improvement of cold-resistance of‘Tunisa soft-seed'by traditional breeding techniques has been constrained by complex biology and long period. Genetic engineering as a supplementation for traditional breeding has been proven to be an effective approach for creating novel germplasms with elevated stress tolerance. Establishment of an efficient and stable regeneration and transformation system is basis of genetic engineering. However, in the pomegranate transformation system, the regeneration of adventitious buds was limited due to the influence of Agrobacterium tumefaciens, antibiotics and other factors involved in the transformation process. Moreover, the induction of adventitious roots was also difficult. In this study, in vitro grafting method was attempted to establish an efficient pomegranate test-tube grafting system in order to solve the problem which the regenerated adventitious buds were difficult to regenerate adventitious roots and ultimately obtain the whole transgenic plantlets.【Methods】In this experiment, the seedlings obtained from seeds of‘Tunisa soft-seed'and‘Yudazi'were used as experimental materials. Different combinations of rootstock and scion were conducted to test the effect on the rate of the grafting survival. The seedlings of‘Yudazi'as rootstocks were divided into two types, including with and without cotyledons. While the seedlings of‘Tunisa soft-seed'as scions were divided into four types, including without leaf, two-leaf, four-leaf and six-leaf tips. Stem tip without leaf blade was as control. In order to obtain the optimal mixture of hormone in MS for pomegranate tube grafting in vitro, the grafted seedlings were then placed in MS supplemented with different concentrations of 6-Benzylaminopurine (6-BA) and 0.1 mg·L-11-Naphthaleneacetic (NAA) . The grafted-seedlings were transplanted and acclimatized. The survival rate of grafting and transplanting were analyzed, respectively.【Results】In the study of the effects of different treat combinations of stock and scion on survival rate of grafting plants in vitro, the results showed that the survival rate of grafting with the stem tip with leaf blade was higher than without leaf blade. The survival rate of grafting was increased with increasing the number of stem tip with leaf blades. And the highest survival rate of grafting was obtained when the number of the scion attached blades was four, then the survival rate of grafting decreased with the increasing of the number of blades in both two types of rootstocks. When the scion was the four-leaf stem tip type, the survival rate of grafting with non-cotyledon rootstock was obviously higher than that of cotyledon rootstock. Therefore, the optimal combination of pomegranate tube grafting which the survival rate of grafting was reached 73.33% was to use the stem tip with four blades as scion and the seedlings without cotyledon as rootstock. The effects of 6-BA on survival rate of grafting was significantly different in different treatments. The results indicated that the survival rate of grafting with 6-BA added to the MS medium was higher than the culture medium without 6-BA. And the survival rate of grafting increased with the increasing the concentration of 6-BA. The highest of the survival rate of grafting was reached 96.67% when 1.5 mg · L^-16-BA was supplemented into the MS medium. And then the survival rate of grafting decreased when 2.0 mg·L^-16-BA was supplemented into the MS medium. The same phenomenon was also observed in the process of acclimatization and transplantation. The survival rate of transplanting increased with the increasing of the 6-BA concentration, and the highest survival rate of transplanting was reached 75.86% when 1.5 mg · L^-16-BA was supplemented into the MS medium. Moreover, the grafted seedlings grew well and healthy. Therefore, the optimal grafting and growth medium after transplanting were both MS supplemented 1.5 mg · L^-16-BA and 0.1 mg · L^-1 NAA.【Conclusion】In this study, the grafting system of pomegranate tube in vitro was established. It provided a new way to solve the transforming adventitious buds difficult to regenerate adventitious roots in the transformation process of pomegranate. Meanwhile, it provided technical support for germplasm improvement through genetic engineering in pomegranate.