Production of Herbicide-Resistant Crops by Cell-Engineering

为农作物开发一种新的选择性除草剂需要很长时间和大量的资金。如果我们能够创造出一种对动物低毒、对环境安全、价格便宜且具有长期实际使用历史的除草剂具有抗性的作物植物，则与为该作物植物开发上述新型选择性除草剂的评价是一样的。在本研究中，研究了几个试验，并作为研究目的的一些可能性的例子，创建抗草甘膦弯曲推杆和抗百草枯向日葵。在抗草甘膦弯黄油的选育中，通过共存除草剂浓度从100 μ m逐渐增加到1000 μ m，获得了对1000 μ m草甘膦具有抗性的愈伤组织。将该耐受性愈伤组织移植到无激素培养基上，在光照下培养。20天后，将生成的植株在1000 uM草甘膦溶液中浸泡8小时。20 d以上，抗性愈伤组织再生植株存活，而感感愈伤组织再生植株死亡。抗性品种的草酸含量为原敏感品种的1 / 6。另一方面，利用电融合系统（SHIMAZU体细胞杂交器，SSH-10）对酶处理后的向日葵和抗百草枯马草叶片原生质体进行融合。在MS培养基（NAA 0.2 ppm, BA 0.5 ppm）上，在25‰的温度下，在3000勒克斯下培养2个月，然后转移到含有百草枯的MS培养基上。在百草枯浓度大于1 × 10^<-5>M时，未融合的向日葵菌落不能形成愈伤组织，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.