Regeneration of plants from cultured guard cell protoplasts

A method of regenerating Nicotiana glauca plants, the steps comprising:

• collecting a leaf sample;
• separating an epidermal layer from said leaf sample under a sterile solution comprising approximately 0.5 % polyvinylpyrrolidone 40 (PVP 40) and 0.5 % ascorbic acid of pH approximately 6.5;
• placing said epidermal layer in a first solution having a pH and comprising approximately:
• 0.2M sucrose;
• 0.7 mM calcium chloride;
• 10.81 g/l Cellulase Onozuka RS;
• 0.081 g/l Pectolyase Y-23;
• 5 g/l polyvinylpyrrolidone 40 (PVP 40);
• and
• 2.027 g/l bovine serum albumin;
• initially adjusting said pH of said first solution to approximately 3.4;
• stirring said first solution for approximately 7 minutes;
• raising said pH of said first solution to approximately 5.5;
• incubating said epidermal layer at 28.degree.-29.degree. C. for approximately 12-15 minutes in an orbital shaking bath at approximately 175 excursions per minute to form a cleaned epidermis;
• collecting said cleaned epidermis;
• rinsing said cleaned epidermis with a solution comprising 0.2M sucrose and 1 mM calcium chloride at an approximate pH of 5.5;
• placing said cleaned epidermis in a second solution having a pH and comprising approximately:
• 0. 25M sucrose;
• 1 mM calcium chloride;
• 16 g/l Cellulase Onozuka RS;
• 0.12 g/l Pectolyase Y-23;
• 5 g/l polyvinylpyrrolidone 40 (PVP 40);
• and
• 3 g/l bovine serum albumin;
• initially adjusting said pH of said second solution to approximately 3.4;
• stirring said second solution for approximately 7 minutes;
• raising said pH of said second solution to approximately 5.5 to form digested epidermis;
• incubating said digested epidermis at approximately 28.degree.-29.degree. C. for approximately 3.25 hours in a reciprocating shaking bath at approximately 15 excursions per minute to form a suspension;
• separating suspended material in said suspension from a flitrate having said guard cell protoplasts;
• centrifuging said filtrate to form a centrifugate;
• pouring off a liquid portion of said centrifugate, leaving a guard cell protoplast mass;
• adding a wash medium to said guard cell protoplast mass, comprising:
• 0.085 gl hydrous calcium chloride (CaCl.sub.2 .multidot.H.sub.2 O);
• 0.088 g/l potassium chloride;
• 1.35 g/l glycine;
• 0.28M sucrose;
• and
• 5 mM of morpholino-ethanosulfonic acid (MES);
• gently suspending guard cell protoplasts of said guard cell protoplast mass in said wash medium;
• centrifuging said wash medium to form another centrifugate;
• repeating said pouring off step, said adding wash medium step, said gentle suspension step, and said centrifugation step, first with a wash medium of pH approximately 6.8, then with two additional wash mediums of pH approximately 6.1;
• growing said guard cell protoplasts in a first medium comprising approximately:
• 0.28M sucrose;
• 0.3 mg/l .alpha.-naphthaleneacetic acid (NAA);
• and
• 0.075 mg/16-benzylaminopurine (BAP);
• said first medium having an approximate pH of 6.1;
• illuminating said guard cell protoplasts with red light of wavelengths approximately between 625 and 800 nm and of fluenee approximately 15-20 .mu.mol m.sup.-2 s.sup.-1 of photons of photosynthetically active radiation (PAR) wherein said red light is cycled on an approximately 12 hour light/12 hour dark cycle;
• transferring said grown guard cell protoplasts to a second medium comprising approximately:
• 0. 3 mg/l .alpha.-naphthaleneacetic acid (NAA);
• 0.075 mg/16-benzylaminopurine (BAP);
• 0.23M sucrose;
• 5 mM morpholino-ethanosulfonic acid (MES);
• and
• 0.5 % agarose;
• said second medium having an approximate pH of 6.1;
• illuminating said second-medium guard cell protoplasts in tightly sealed Petri dishes with continuous white light of approximately 21-27 .mu.mol m.sup.-2 s.sup.-1 of photons of photosynthetically active radiation (PAR) to establish a viable callus cell population;
• transferring said viable callus cell population to a callus medium comprising approximately:
• 0.2 m/l indole-3-acetic acid (IAA);
• and
• 0.2 mg/l kinetin to further establish guard cell callus;
• transferring said guard cell callus to a shoot medium comprising approximately:
• 1 mg/l kinetin to establish shooted guard cell callus;
• illuminating said shooted guard cell callus with continuous white light of approximately 30/.mu.mol m.sup.-2 s.sup.-1 of photons of photosynthetically active radiation (PAR);
• transferring said shooted guard cell callus to a root medium comprising approximately:
• 3 mg/l indole-3-acetic acid (IAA) to form an immature plant;
• and
• planting said immature plant.