Study On Micropropagation Of Chinese Chestnut In Vitro

The objective of the studies is to achieve a reliable protocol of propagating Chinese chestnut (Castanea mollissima BL.) in vitro. At the beginning of this paper, the development of the researches on Plant Tissue Culture (PTC) in the past was reviewed on the whole, mainly focused on those of the Wood Plant (WP). Then the advances of micropropagation of chestnut (Castanea Miller) in vitro both home and abroad were summarized in terms of explants, multiplication, and rooting. Meanwhile, three obstacles including Low Survival Rate (LSR), Vitrification, and Shoot-tip Necrosis (STN), which have happened in many researches before, were also presented.On basis of the reviews, a research on Chinese chestnut's PTC was conducted at the Beijing Forestry University (BFU) from the beginning of 2003. Some achievements have already been made. By comparisions of four explants types, like chestnut axillary bud from 3-month-old seedlings and adult plant, hypocotyls, and cotyledon, using chestnut axillary bud from 3-month-old seedlings and hypocotyls were successfully regenerated and proliferated in vitro. After the subculture for around 2 years, Chinese chestnut had been successfully rooted in vitro. However, the in vitro culture of axillary bud from adult material and cotyledon were not able to be established till present. The details in these regards are as follows:(i) Selection and Disposal of Explants. Axillary buds from 3-month-old-seedlings were sterilized by a quick dip in 75% alcohol followed by immersion for 16 to 18 minutes in 0.1 HgCl containing a few drops of Tween 80. The basal culture medium for initiation was Wood Plant Medium (WPM) with growth regulator benzyladenine (BAP) at the concentration of 1.0 mg/L. The dispodal of hypocotyls was as same as that of axillary buds but immersion for 13 to 15 minutes, Murashige and Skoog's medium with BAP at the concentration of 2.0mg/L was appropriate.(ii) Shoots-multiplication. The basal medium for shoot-multiplication was WPM. By quadratic regressive orthogonal device for the choice of hormone, the best combination and concentration was BAP (1.25 to 1.53mg/L) with indolebutyic (IBA, 0.08 to 0.15mg/L). Other conditions should be sugar (30mg/L), pH (5.8). The microcuttings were subcultured on a 6-week-long cycle.(iii) Shoots-elongation. The WPM with BAP (0.3mg/L) was employed in the process of shoots-elongation. It has been proved to be efficient.(iv) Rooting. After the subculture for 7 to 9 generations, the Chinese chestnut was rooted in vitro. There were two ways to induce roots. One was that the microcuttings were cultured on the 1/4MS with IBA (1mg/L), 40% rooting rate was made. Callus and shoot-tip necrosis were happened in this way. The other was that the microcuttings were cultured on 1/4MS with IBA (3mg/L) at dark condition for 5 days, and then transfer to the 1/4MS with auxin-free, some roots were obtained with higher quality.