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以陆地棉（ＧｏｓｙｐｉｕｍｈｉｒｓｕｔｕｍＬ．）栽培品种“鲁棉６号”下胚轴的胚性愈伤组织为材料，制备并培养原生质体。采用继代培养７～９ｄ、活力旺盛的胚性愈伤组织，在１％纤维素酶、１％果胶酶、０７ｍｍｏｌ／ＬＫＨ２ＰＯ４、２５ｍｍｏｌ／ＬＣａ２＋、０５ｍｏｌ／Ｌ甘露醇、ｐＨ５８、３０℃的条件下，具活力的原生质体得率最高。经分离纯化后，原生质体在含有０４５ｍｏｌ／Ｌ葡萄糖的Ｋ３无机盐、ＮＴ有机物并附加０１ｍｇ／Ｌ２，４Ｄ、０２ｍｇ／ＬＫＴ的培养基中静置１０～１５ｍｉｎ，原生质体分成上下两层。下层多为质浓、内含物丰富的小圆球形原生质体，在培养过程中易于分裂并形成愈伤组织。棉花原生质体在上述培养基中培养３ｄ后出现第一次分裂，２～３周后形成小细胞团，此时需添换低渗培养基１～２次，小细胞团才能持续分裂并形成愈伤组织。再生愈伤组织块达３～５ｍｍ时，转入固体培养基繁殖、分化胚状体并形成再生植株。实验表明，采用活力旺盛的胚性愈伤组织及适时添换低渗培养液是棉花原生质体成株的关键。
The embryogenic callus of hypocotyl of Lintan 6 under Gosypium hirsutum L. was used as material to prepare and cultivate protoplasts. The embryogenic callus with subcultured vigorously for 7-9 days was cultured in the medium of 1% cellulase, 1% pectinase, 07mmol / L KH2PO4, 25mmol / L Ca2 + and 05mol / L mannitol, Under the conditions of pH58 and 30 ℃, the viable protoplasts yield the highest yield. After isolation and purification, the protoplasts were kept in the medium of K3 inorganic salts containing 0.45mol / L glucose, NT organics and added with 0.1 mg / L, 4 D, 0.2 mg / L KT for 10-15 min, and protoplasts Body is divided into two layers. The lower is mostly thick, rich in content of small spherical protoplasts, easy to divide in the culture process and the formation of callus. The protoplast of cotton was cultured in the above medium for 3 days and then the first division occurred. After 2 ~ 3 weeks, the small cell mass was formed. At this time, the low-permeability medium was added 1 ~ 2 times, the small cell mass could continue to divide and form more Wound tissue. Regenerated callus pieces up to 3 ~ 5mm, transferred to solid medium propagation, differentiation embryoid body and the formation of regenerated plants. Experiments show that the use of vigorous embryogenic callus and timely replacement of low-permeability culture medium is the key protoplasm cotton plant.