Production of Herbicide-Resistant Crops by Cell-Engineering

开发一种新的作物选择性除草剂需要很长的时间和大量的资金。如果我们能够创造出一种对动物低毒、对环境安全、价格低廉、实际使用历史长的除草剂具有抗性的农作物，那么它就被评价为与开发上述针对此类作物的新型选择性除草剂的事情是一样的。在本研究中，考察了几个试验，作为具有一定研究目的的例子，创造了抗草甘膦的弯曲推杆和抗百草枯的向日葵。在选择抗草甘膦的弯曲黄油时，将共生除草剂浓度从100微米逐渐增加到1000微米，获得了抗草甘膦1000微米的愈伤组织，并将其移植到无激素的培养基上，在光照下培养。20天后，将产生的植株在1000um草甘膦溶液中浸泡8小时。20d以上，抗性愈伤组织再生的植株存活，而敏感愈伤组织再生的植株死亡。用酶法制备的向日葵和抗百草枯原生质体经岛津体细胞杂交仪(SSH-10)电融合系统进行融合。融合子在MS(NAA0.2ppm，BA0.5ppm)，25ﾟC，3000lux下培养2个月后转移到含有百草枯的MS培养基中。未融合的向日葵菌落不能形成1×10~(-5)~(-5)M百草枯的愈伤组织，在此条件下融合细胞可以形成愈伤组织和再生植株。

Long time and much money are required for the development of a new selective herbicide for a crop plant. If we can create a crop plant having resistance to a herbicide which is low-toxic to animals, safe to the environment, cheap and has a long practical use history, it is evaluated as the same matter as the development of above-described new selective herbicides for such crop plant.In this study, severaltrials were examined, and as the examples having some possibility for the research purpose, the creation of glyphosate-resistant bent putter and paraquat-resistant sunflower.In the selection of glyphsate-resistant bent butter, callus having resistance to 1,000 uM glyphosate were obtained by the gradual increasing of the concentration of co-existing herbicide from 100 until 1,000 uM.This tolerant callus was transplanted to hormone-free medium and cultured in the light. After 20 days, the generated plants were soaked in 1,000 uM glyphosate solution for 8 hours. After more 20 days the plant regenerated from resistant callus were survival while ones generated from susceptible callus were dead. The sikimic acid content of the resistant one was one sixth of original susceptible one.On the other hand, both protoplasts prepared from leaves of sunflower and paraquat-resistant horseweed by enzyme treatment were fused by a electrofusion system (SHIMAZU SOMATIC HYBRIDIZER,SSH-10). The concentration of both protoplasts was 100 thousands cells per ml. The fusates were cultured on MS medium (NAA 0.2 ppm, BA 0.5 ppm), at 25ﾟC under 3,000 lux, for 2 months and moved to paraquat-containing MS medium. Unfused sunflower colonies could not form any callus upper 1 x 10^<-5>M paraquat, under which condition the fused cells could form callus and regenerated plants.