Establishment and application of callus induction and suspension culture system of pear anther

Abstract: 【Objective】Pear is one of the important fruit species in the world，and pear growing has become an important pillar industry in many places for its huge economic value，which plays a significant role in farmers’income and social stability. Pear’s genome is highly heterozygous, which is resulted from cross-pollination, bringing serious difficulties to the genetic transformation. Cell suspension culture is an important culture method for cells. It can not only provide raw materials for protoplasm preparation, regeneration, genetic transformation and somatic cell hybridization, but also be used for the production of secondary metabolites. In this paper, we firstly obtained calluses by inducing the anthers of Xinli No.7 pear and then studied effects of culture medium types and hormone combinations on the growth of pear callus under suspension culture conditions by selecting the loose calluses from the vigorous growths after induction as materials, in order to establish a suitable suspension culture system for pear anther calluses, obtain pear calluses of high quality and provide good materials for pear protoplast preparation and pear genetic transformation.【Methods】Pear anthers were taken as the callus inductionmaterials. Flower buds at the“small-balloon stage”(transverse diameter = 2.7-3.0 mm, longitudinal diameter = 8.0-8.5 mm) of Xinli No.7 pear trees were selected, cleaned and soaked in 75% alcohol for 25 s and 0.1% HgCl2 for 8min successively, and then the petals were removed and the anthers were taken out. The anthers were cultured on the induction medium (1/2 MS + 1.0 mg · L- 1 2, 4-D + 0.4 mg · L^{- 1} NAA + 30 g · L^{- 1} sucrose + 7 g · L^{- 1} agar powder, pH 5.8-6.0) for 30 days. After several subcultures on the medium (MS + 1.5 mg  · L^{- 1} 2,4-D + 0.5 mg  · L^{- 1} 6-BA + 30 g  · L^{- 1} sucrose + 7 g · L^{- 1}  agar powder), loose calluses were selected for the suspension culture experiments, in which the basal medium was MS + 30 g·L-1 sucrose (pH 5.8-6.0), and the suspension medium was prepared by adjusting the concentration of 2, 4-D (1.0, 1.5, 2.0 mg·L-1 ) and 6-BA (0.5, 1.0, 1.5 mg · L^{- 1} ) in the basal medium. Then 1.5 g calluses were put into 40mL liquid suspension medium and cultured for 8 days (28 ℃, 200 r·min^-1 , dark) as for the initial culture. The calluses were subcultured every other 7 days, and the growth quantity and mortality of suspended calluses were measured after 28 days. In order to verify whether the callus obtained by suspension culture could be used as genetic transformation material, agrobacterium GV3101 solution containing pROK2-GFP plasmid (OD600=0.5) was used to infect the calluses for 15 min. Subsequently, they were cultured on the symbiotic medium (MS+30 g · L^{- 1}  sucrose+1.5 mg · L^{- 1} 2, 4-D +1.0 mg  · L^{- 1} 6-BA+ 7 g · L^{- 1} agar powder, pH 5.8-6.0) for 3 days, transferred to the screening medium (MS+30 g·L-1 sucrose+ 1.5 mg · L^{- 1} 2, 4-D+1.0 mg · L^{- 1} 6-BA+20 mg · L^{- 1} Basta + 300 mg · L^{- 1} Cef + 200 mg · L^{- 1} Tim + 7 g · L^{- 1} agar powder, pH 5.8-6.0) for 20 days, and the transformation efficiency was calculated. Transformation efficiency (%) =number of green fluorescent calluses/total number of infected calluses ×100. In order to identify whether or not the suspension cultured calluses (cells) could be used to extract high-quality protoplasts, we collected the stable suspension cell lines on the 4th day by centrifuging them at 6 000 r·min-1 for 5 min. Then we split them in the lysis buffer for 10 min at 55 ℃. 10 mL lysis buffer contained 1.0% cellulase, 0.5% macerozyme, 1 mL MES pH5.7 (20 mmol · L^{- 1} ), 5 mL mannitol (0.5 mol · L^{- 1} ) and 2.5 mL KCl (20 mmol · L^{- 1} ). Thereafter, we cooled down the splitting mixture to the room temperature, and added 10 mmol · L^{- 1} CaCl2 and 0.1% bovine serum albumin (BSA) into it. Protoplasts were obtained by filtrating the mixture with a 0.45 μm filter. Moreover, the yield and vitality of the protoplasts were determined.【Results】The induction rate of calluses induced from Xinli No.7 pear anthers during the“smallballoon stage”was 45.80%. When the concentration of 6-BA was constant, the status of callus structures ranged from compact to lose and the status of calluses became better with the increase of the concentration of 2, 4- D. After 4 to 5 times of subculture, yellow and white granular and loose calluses could be obtained. Then the obtained loose calluses were cultured on the suspension medium under 28 ℃ and dark conditions, and after 4 to 5 times of subculture suspension, cell lines were gained. When the concentration of 2, 4-D was 1.5 mg ·L-1 and 6-BA was 1.0 mg · L^{- 1} , the living cell rate and the nearround cell rate reached 81.98% and 83.66%, respectively. Both of them were significantly higher than that of the control, and the suspension mixture was milky white and very stable. Therefore, the optimal suspension culture medium for pear anther calluses was MS+1.5 mg · L^{- 1} 2, 4-D+1.0 mg · L^{- 1} 6-BA + 30 g · L^{- 1} sucrose (pH 5.8-6.0). The growth of pear suspended cells showed a sigmoid curve. From 0 to 2 days, the suspension cells were in the lagging-growing phase, the growth increment was quite low and the cell mortality ranged from 13.97% to 22.50%. From 2 to 4 days, the suspension cells were in the logarithmic-growing phase, the volume, the fresh weight and the dry weight of them increased significantly and the cell mortality was the lowest, ranging from 13.97% to 15.40%. From 4 to 6 days, the suspension cells were in the plateau-growing phase, the growth increment of them was stable, and the cell mortality ranged from 15.40% to 19.14%. In addition, in this stage, the fresh weight was about 0.22 g andthe dry weight was about 0.05 g, both of which reached the maximum value. After 6 days, the suspension cells were in the senescence-growing phase, the growth increment of them decreased and the cell death mortality ranged from 19.14% to 19.18%. Simultaneously, it was observed that a thick layer of senescent cells deposited at the bottom of the bottle, which was adverse to the growth of suspended cells. It was obvious that the survival rate of the suspension cells was all above 77.50% during one subculture cycle of 0-8 d, and the cells had the fastest growth rate and the highest activity during 2-4 d. After the suspension culture, calluses were moved onto the solid medium. They also had the fastest growth rate and the highest activity, and could propagate to the required amount within 15-20 days, which was significantly faster than that of the calluses without suspension culture. Moreover, the transformation rate of calluses with suspension culture was 20.00%, the yield and the vitality of protoplasts separated from the calluses was 3.67×106 per milliliter and 92.00%, respectively.【Conclusion】A suspension culture system suitable for pear calluses was established, and pear calluses of high quality were obtained by this suspension culture system. The pear calluses could be used as materials for genetic transformation or protoplast preparation.