Beijing Agricultural University

The culture and transformation of protoplasts

--Sai Jiqing, Liu Zhihua, Ding Qunxing and Xie Youju

Protoplasts were isolated and purified from BMS (Black Mexico Sweet) suspension cells by different methods. The results show that the direct filtering method can give the highest yield of 3-4 x 106 protoplasts per gram fresh weight and that collecting protoplasts on the interface between KMC solution (Theor. Appl. Genet. 53:57, 1978) and 0.6M sucrose solution can enhance the level of protoplast purification even though the protoplast yield reduces.

The protoplasts were cultured in modified NN67, N6ap, KM media. The culture methods included thin liquid layer, multiple layers, embedding, C. A. Rhodes method (Bio/Technology 6:56, 1988) and so on. The highest plating efficiency of 3.5 x 10-3 could be obtained when 0.45M glucose was used as the stabilizer of osmotic pressure and the protoplasts were embedded in modified N6ap or KM medium with 0.6% low melting point agarose.

A special vector was constructed for protoplast transformation: Plasmid-like DNA S1 was purified from S male sterile cytoplasmic mitochondria of maize. Almost the full-length of S1 was cloned to pBR322 and the recombinant plasmid was named pBS. The GUS (glucuronidase) reporter gene with CaMV 35S promoter from pBI221 was inserted into plasmid pBS and two kinds of recombinant plasmids (pBSG1 and pBSG2) were obtained due to reversal direction of integration (Fig. 1). Increasing transformation frequency was hoped for this kind of vector construction due to homology between plasmid-like DNA S1 and maize nuclear DNA (Nature 304:744, 1983).

With the success of protoplast culture and vector construction, the Baekon 2000 Advanced Gene Transfer System was used to transfer pBSG or pBI221 (CK) containing 35SP-GUS-Nt chimeric gene into protoplasts. Suitable electroporation parameters were studied before doing transformation. In the experiments, the burst time (TB), the number of cycles (CY) and the distance between the positive electrode and the surface of gene-cell mixture (D) were stabilized. The amplitude (A) and the number of pulses (NP) were changed to observe the effects of the different combinations of these two parameters on the frequency of protoplast division. The results are presented in Table 1.

Table 1. Effects of different combinations of electroporation parameters (A and NP) on the frequency of protoplast division (%).
 

		A(KV)
NP	2	6	10
22	32.01	33.00	32.02
24	30.39	32.69	24.50
26	34.96	30.06	22.87

The frequency of protoplast division of control (without electroporation) was 32.67%; T_B=0.4s, C_Y=10, D=2mm; Electroporation: No. protoplasts/ml=2 x 106, volume of electroporation mixture=200ul; Culture: No. protoplasts/ml=5 x 10⁵; The Nos. in the table are the means of 5 fields of vision of inverted microscope from 3 experiments 10 days after culture.

When the amplitude was increased to 10KV and the number of pulses to 2⁴ or 2⁶, the frequency of protoplast division reduced obviously. We thought that the parameters A and NP suitable for maize protoplast transformation should be a compromise between increasing transformation frequency dependent on raising these two parameters and reducing the protoplast division frequency. So the parameters A and NP we used were 10KV and 2⁴. The stabilized parameters T_B, C_Y and D were 0.4s, 5 and 2mm respectively in each experiment. The clones (colonies) from electroporated protoplasts growing to 1-2mm in diameter were picked onto N6ap medium in relatively low density. When the calli from colonies were approximately 1-2cm in diameter, the GUS gene expression was assayed using the histochemical method (Bio/Technology 6:559, 1988). The number of blue calli were counted (Ed. note: color print has been provided and will be supplied on request). The results are in Table 2.

Table 2. Transformation frequencies by electroporation.
 

Plasmid	No. calli assayed	No. blue calli	Percentage of blue calli1	Absolute transformation frequency2
pBSG	201	23	11.44	5.49x10-4
pBI221(CK)	168	17	10.12	1.82x10-4

¹This is also the relative transformation frequency.

²Equal to relative transformation frequency x plating efficiency.

No significant difference of the relative transformation frequency between pBSG and pBI221 (CK) as foreign DNA can be seen from Table 2. The reason for this may be that the size of pBSG is much bigger than that of pBI221. Further studies are underway.

Figure 1. Construction of recombinant plasmids pBSG1 and pBSG2.

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