Some Orchids Conservation In Vitro

Orchidaceae is an enormous family, many Orchids are significant ornamental and ornamental plants. All wild orchid plants were put on a list of CITES(Convention on International Trade in Endangered Species of Wild Fauna and Flora) in 1975. For the past few years, because of human factors, wild orchids sharply reduced, many disappeared.orchids conservation is extremely urgent. Now ways to protect orchids are too single, and status of orchid plants is worrying in China. Orchids conservation is in urgent need of various technologies.This paper studied on Dendrobium densiflorum, hybrid of Dendrobium, Phalaenopsis hainanensis and Dendrobium aduncum, estsablished technological procedure of conservation in vitro, and provided technical support for their germplasm conservation. It also hoped to provide reference fo other orchids conservation.The main results are as follows:1. Aseptic germination procedure, cryopreservation procedure of pollens, seeds and PLBs ofDendrobium densiflorum were established.①Procedure of aseptic germination:after artificial pollination for 4 months, Dendrobium densiflorum capsules were treated with 75% alcohol for 30s, then immerged into 0.1% HgCl2 for 10min. 1/4MS+100g/Lbanana was used for seeds germination, 1/2MS+NAA2mg/L+50ml/Lcoconut water for rooting, and sphagnum moss for transplanting.②Procedure of pollens cryopreservation:fresh pollens(moisture content 43.26%) were immersed into LN without desiccation, and thawing by room temperature, running water or warm water(37℃) after cryopreservation. Pollens after cryopreservation can germinate in vitro normally, and the percent of pollen germination didn't decrease. These pollens can produce capsules after artificial pollination, seeds in capsules can produce vigorous plants.③Procedure of seeds cryopreservation:dried seeds (moisture content 2.78%) were immersed into LN, and thawing by running water for 30 min after cryopreservation. Vitality of seeds can reach to 66.76%, a little more than fresh seeds. These seeds germinated normally in 1/4 MS+100g/L banana, and produced vigorous plants finally.④Protocorm cryopreservation procedure was preliminarily established. After germination for 60d, protocorms were pre-cultured for 4 days in MS culture medium supplemented with 0.5M sucrose at 25±2℃, treated in Loading solution for 50min and in PVS2 vitrification solution at 0℃for 50min. Then protocorms were directly plunged into liquid nitrogen. After rapid thawing in a water bath at 37℃for 60s, washed in Unloading solution for two times, each time for 10 min. Vitality of protocorms after cryopreservation can reach to 98.04%, and protocorms can transfer to 1/4MS+100g/L banana in dark for 3 weeks. PLBs can regenerate after exposuring to light for 7 weeks.Aseptic germination and cryopreservation of seeds procedure of Dendrobium densiflorum can use to Conservate the same plant materials of hybrid of Dendrobium in vitro. After cryopreservation, vitality of seeds can reach to 68.11%, a little more than fresh seeds. These seeds can germinate normally and produce vigorous plants finally.2. Procedure of ordinary temperature conservation of Dendrobium aduncum was established.At first, stems were treated with 75% alcohol for 30s,0.1% HgCl2 for 10 min. MS+6-BA 2.0mg/L+NAA0.1mg/L was used for inducting cluster buds, MS+6-BA2.0 mg/L+NAA0.5 mg/L for bud multiplicating, MS+NAA0.5 mg/L+AC 2g/L for rooting, and Sphagnum moss for transplanting. 3.Aseptic germination and regeneration procedure of Phalaenopsis hainanensis in vitro were established.After artificial pollination for 4 months, Phalaenopsis hainanensis seeds were sowed in 1/4MS+100ml/L coconut water. 1/2MS+6-BA2.0mg/L+NAA0.5mg/L+100g/L banana was used for multiplication, 1/2MS+6-BA0.5mg/L+NAA2.0mg/L+100g/L banana for differrentiation, MS+NAA0.1mg/L+100 g/L banana for rooting, and sphagnum moss for transplanting.