Primary Study On Regeneration System Of Phyllostachys Heterocycla Var.pubescens

Phyllostachys heterocycla var.pubescens is the most important bamboo species in China, it covers the largest area and takes the highest economic value among all bamboos. The recombination of genetic resources is difficult because of its special biological characteristics in flowering, which leads to an infeasibility on oriented-genetic-improvement. Transgenic techniques in other plants has made a great advancement, and the rapid growth of bamboo section nowadays needs urgently germplasm innovation to improve its genetic elements, so an exercised generation system on bamboos is necessary which will made it possible for bamboo transgenic breeding.By using bamboo shoots, embryo and other organs as explants, a serial of works on generation system of Phyllostachys heterocycla var.pubescens were conducted, i.e., disinfection of explants, callus induce and reproduction, differentiation and suspension culture, cellular observation, and the results were:1. Disinfection of bamboo shoots (rhizome-shoots, spring-shoots, winter-shoots) was easy, by using bamboo shoots as explants, a 95% of disinfected explants can be got after applying 75% alcohol, only a few were contamination occasionally.Disinfection of embryos was rather difficult, disinfections had different effect on bamboo seeds: Ca(ClO)2 had great damage on seed germination, AMP was somewhat valid to fungi on seeds, H2O2 was somewhat noneffective, 0.1% HgCl2 was an effective disinfectant. The sequence of disinfection was as follows, firstly, the bamboo seeds were pre-dealt, and tips on the other end of the embryo were often cut down in order to decrease infection rate, after dipping into water for 12h, then they were disinfected for 20min in 0.1%HgCl2, at last, they were washed 10 times by using distinfected water, thus good distinfected explants can be got. By this way only a few explants were infected by singled fungi, bacteria and other microorganisms were hardly found, so it's supposed that the prominent fungi on seed were the vital infectant.Bamboo seeds of more than one–year-old were not used as explants for extremely low germination; and if the seeds were picked before physiological maturity, the contamination rate decreased along with a low germination. Warm water made damage on seeds vigor and had no help on disinfection.2. All shoots (rhizome-shoots, spring-shoots, winter-shoots and sheath) can be induced callus easily, but those callus were not in good condition and no close embryogenic callus existed, all callus were highly watered and browning seriously during subculture, with no amelioration under any remedying measurements, most of those callus were absent with karyon under cellular detection. Different putting ways and cuttings had no effect on the indumenta of callus. Seed part which touched on culture medium was easier to induced callus; as for induce time, winter-shoots was fastest, rhizome-shoots was the middle, and the winter-shoots needed the longest time.The callus induced from embryo had two types, one was closed embryogenic callus, and the other was loose non-embryogenic callus. Cutting ways worked on the callus, crosscut was relatively helpful in induction of embryogenic callus because of it cut down the root parts which were easy to induce non-embryogenic callus. As for putting ways, putting by scutellum downward would got a high callus rate, while putting by the adverse way would decrease the callus. Embryogenic callus often generated from root-stem junctions or scutellum, yellowish, touched closely by nippers; non-embryogenic callus usually produced from root, white or dark brown, loose, soft, brittle and highly watered.3. Callus induced from bamboo shoots (including spring-shoots, winter-shoots, rhizome-shoots and their sheath) browning seriously on differentiation culture medium, with no differentiation under all kinds of situation, and couldn't be reproduced..Only a few callus induced from bamboo embryo could be differentiated but got no plantlets; browning happened seriously on embryogenic callus with a few could be reproduced occasionally, those mbryogenic callus grew slowly and not sensitive to all kinds of culture situation, whether subculture or not, they were developed to brown and death gradually.As for survival of callus, organic additive such as praline, glutamine, casein hydrolysate and coconut water except yeast extract could all improve the survival rate, while shoots milk additive would give a reverse rate. Kinds of carbohydrate had little effect on callus induce, glucose, sucrose, fructose, maltose, lactose except mannose had hardly no different on the results. Culture medium types also had no different function on callus culture such as MS,CC,WC,NB,KM8P, only N6 had a relatively low effect.4 Suspension culture of non-embryogenic callus showed that they were insensitive to 2,4-D concentration, those callus began a relatively rapid proliferation rate after cultured 12d in liquid medium. Increase of inoculation amount could promote proliferation some degree but with no certain relation. The results demonstrated that the non-embryogenic callus were not sensitive to culture situation and proliferating slower under suspension culture.5 By cellular observation, callus induced from shoots (spring-shoots, winter-shoots and rhizome-shoots) had hardly any karyon, single cell was large, long and a little curved, with a loosely arrangement. It was supposed that lack of karyon maybe the reason of their inability of proliferation.Two types of callus induced from seed were different, cells in closely embryogenic callus were small with large karyons and small vacuoles, with a dense arrangement; while cells in non-embryogenic callus were big with a few karyon and arranged loosely.