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IV. Protoplast Isolation, Culture, and Genetic Manipulation

IV. Protoplast Isolation, Culture, and Genetic Manipulation

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HU HAN AND SHAO QIQUAN



B. PROTOPLAST

CULTURE



Plants have been regenerated successfully from protoplasts of tobacco, petunia,

dnd carrot (Li et al., 1978a,b; Wu et al., 1977). Efforts to generate plants from

protoplasts derived from cereals, legumes, and other species have been less

successful (Yen and Li, 1979; Li et al., 1978a,b; Cytology Lab., 1977; Tsai et

al., 1978).

Recently a two-layer culture method, i.e., liquid in the upper layer and solids

in the lower, has been developed, and plants have been regenerated from

mesophyll protoplasts of Nicotiana rustica x N . alata (Hsia et al., 1979). Moreover, typical division of cells regenerated from wheat and barley mesophyll

protoplasts has been observed at about 0.1% frequency (Li, 1979a,b).

Of the nutrients explored, vitamin and other organic compounds are very

important to protoplast division, while glucose and low levels of other sugars

have favorable effects on wheat protoplast culture. When protoplasts were cultured under lower osmotic conditions, e.g., 0.4 M glucose, budding, bulbing,

and anuclear subprotoplasts were generated probably due to rapid swelling of

protoplasts and incomplete cell wall formation (502 Group, 1974).

More recently somatic hybrid plants have been regenerated through fusion of

protoplasts from somatic cells of N . tabacum and those of N . rustica. Examination of chromosome number and identification of peroxidase isoenzymes revealed that the plants are of somatic hybrid origin (Wang et al., 1981; Gong

et al., personal communication).

C . GENETICMANIPULATION



Homologous fusion of mesophyll protoplasts of wheat, corn, and other species,

and heterologous fusion of those of wheat and Viciafaba by adding NaN03 to

culture medium were first obtained at a low frequency of approximately 4%

(Sun, personal communication). Using Kao’s fusion technique with high pH,

calcium ion solution, and PEG,interspecific fusion was induced between protoplasts from wheat yellowing leaves and those from green leaves of petunia. The

fusion occurred at a frequency of 25%, and 10% of the fusion bodies were of

heterologous origin. Nuclear staining confirmed that the fusion bodies were

heterokaryons. Some of the fused protoplasts regenerated cell wall, underwent

cell division, and developed into small calli after being transferred to fresh

medium, but they could not be identified as hybrids due to lack of a selection

system.

In chloroplast transplantation research, chloroplasts of wheat and spinach were

introduced into carrot callus protoplasts with PEG used as inducing agent and



PLANT CELL AND TISSUE CULTURE IN CHINA



9



successful transfers occurred at a frequency of 2-5% (C. Ma, personal communication).

The characteristics of rape mosaic virus (YMV 15) were studied by introducing

the virus into tobacco (N. tubacum var. Sumsan) mesophyll protoplasts. It was

found that YMVl5 is serologically related to TMV, but the infection pathway is

different. When polyomithine was added, 46-96% of tobacco protoplasts were

infected by YMVI5, and each infected protoplast contained (1-3) X 105 virus

particles (Tian et al., 1977).

Crown gall cell is useful in plant genetic engineering. In general plant tumor

cells induced by different Agrobacterium tumefaciens strains cannot grow on

medium containing o-lactose or D-galactose as the sole carbon source, since

both sugars are toxic to these tumor cells. However, Li Xiang Hui and

Schieder ( 1981) recently discovered that tobacco tumor cells induced by

A . tumefaciens strain B6S3 are different from other tumor cells and can

grow on medium supplemented with D-lactose as the sole carbon source.

This characteristic might provide a selective system for protoplast fusion

and transformation experiments. Somatic hybrids have been obtained through

fusion of protoplasts from B6S3 tobacco tumor cells and those from mesophyll

of N. tubacum cv. Xanthi. The hybrid characteristics were demonstrated by the

presence of octopine identified in the hybrid cells. It now appears possible to

transfer T:DNA segments of the tumor cells to normal ones through protoplast

fusion (X. H. Li, personal communication).



V. SELECTION OF MUTANTS

Cell cultures are also used as means of obtaining variant strains. Investigations

on selection of mutants at plant cell levels got under way recently in China. This

appears to be a promising field of research.

A chlorophyll mutant of rice (HY 101) was obtained that segregated at a

frequency of 19% from an H2strain. The anther callus of variety No. 8126 was

treated with ethyl methane sulfonate (EMS) at an early stage in culture (Hu er

al., 1981) and the aforementioned H 2 strain was obtained from this callus. The

mutant plants are yellowish-green in color and stable in characteristics. The

reciprocal F, s obtained from crosses between the mutant and normal rice plants

were green. The F2population segregated in a 3 : 1 ratio of green yellowish-green

plants. Green and yellowish-green plantlets appeared at a ratio of 1 : 1 in a plant

population obtained from hybrids through anther culture.

Chen et al. (1980a) were able to obtain a BUdR-resistant mutant from soybean cell line SB-1 which was irradiated by 1000-R y rays, and cultured for 20

days on y-rayed B5 medium. It is believed that irradiation might trigger chemical changes in culture medium in addition to the direct effects on the treated cells.



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HU HAN AND SHAO QIQUAN



Thus the mutation rates may be increased by exposing the calli to the direct and

indirect irradiations.

Ho et al. (1980) were able to select a lysine analog (0L)-resistant tobacco

callus mutant at a low frequency (ca. lo-') by first subculturing tobacco (N.

tubacum L. cv. Gexin No. 5 ) callus through three successive passages on a

medium containing lg/ 1 L-Coxalysine (OL), an inhibitor of callus growth, then

culturing the treated tissue through 6 passages on a medium without the selective

agent OL, and finally treating the callus cultures with 2% EMS. The resultant

mutant is 10 times less sensitive to OL than the parent line, and in general it was

stable through 12 passages of subcultures in the absence of the selective agent. It

was found that the lysine analog-resistant tobacco callus mutant has accumulated

twice the normal level of lysine content, while a different pattern of peroxidase

isoenzyme spectrum has been discovered electrophoretically in the mutant as

compared with that of parent lines.



VI. MISCELLANEOUS: IN VITRO PROPAGATION

THROUGH PLANT TISSUE CULTURE

A number of economically important plants, including seaweeds, have been

propagated through tissue culture in China. In addition, many drug-producing

plants have also been cloned in vitro.

A. TISSUE

CULTURE

OF DRUG-PRODUCING

PLANTS



Peking Pharmaceutical Institute was able to cultivate ginseng callus proliferated from young stems and roots. It was found that the total ginseng saponins was

the same as that of the garden ginseng. For example, the ginseng callus contains

4% (dry weight) saponins, whereas the 6-year-old garden ginseng contained 3%

(dry weight) of this chemical. Zheng and Liang (1976) were able to cultivate the

famous herb Panax noroginseng, and chemical analysis showed that the herb

contained 10.25% (dry weight) of gross saponin and sapogenin, in contrast to the

tuberous roots of the natural plants, which contain 6.06% (dry weight) of the said

drugs only. It has been found that callus tissue of Seopolia ocutagula plant

produced 0.55% (dry weight) of Hyocyamine and seopolamine, whereas fieldgrown plants had only 0.139% of these drugs (Zheng and Liang, 1977).

B. In Vitro PROPAGATION

BY PLANTTISSUE

CULTURE



Since 1975, Wang et al. (1975a,b; Wang and Chang, 1978a,b) have successfully regenerated seedlings from embryo (diploid 2n = 18) and endosperm



PLANT CELL AND TISSUE CULTURE IN CHINA



11



callus (triploid 3 n = 27) of Citrus sinensis. The diploid plantlets have been

easily induced either from bud primordia or through embryoid formation. The

triploid seedlings were regenerated through embryogenesis of endosperm calli,

which had been induced at the cell stage.

A successful system for obtaining “virus-free’’ seed potato has been worked

out by a number of institutions. Virus-free potato seed is now successfully

produced and planted in about 20 provinces. The yield has increased significantly

through the use of ‘‘virus-free’’ seed (unpublished results).

Clones of sugarcane have been obtained through tissue culture in all canegrowing provinces (unpublished results). Meristem of stem apex or lateral buds

as well as young leaves are used as initial culture materials for the induction of

callus from which young seedlings are regenerated. The yield of canes raised

through “test-tube” methods is the same as that of the control, but the tissue culture-obtained clones are more variable as some have more tillers and others have

smaller stems.

I n vitro fertilization of corn ovules was first achieved in 1977 with a simplified

medium prepared mainly from natural extracts of potato (Shao et al., 1977).

Fourteen seeds were produced in 1978, and the portion of seed set was 0.42%.

Hybrid kernels matured in about 20-22 days after pollination and germinated in

a test tube. After transferring to pots, only two plants survived. One of them had

clear purple markers on leaves and stem. The chromosome number of root cells

was 20. These results indicated that plants produced from test-tube fertilization

were intervarietal hybrids but not parthenogenetic haploids (Jiang et al., 1979).

ACKNOWLEDGMENTS

The authors wish to thank H. W.Li for his help in preparing the manuscript and R. B. Tan for her

technical assistance.



REFERENCES

D’Amato, F. 1978. In “Frontiers of Plant Tissue Culture” (T. A. Thorpe ed.), pp. 287-295.

University of Calgary Offset Printing Office, Calgary, Alberta, Canada.

Breeding Laboratory, Heilungchiang Sugarbeet Institute. 1973. In “Proceedings of Symposium on

Anther Culture,” pp. 304-305. Science Pxess, Peking.

Chen, C. H., Chen, F. T., Chien, C. F., Wang, C. H., Chang, S. J., Hsu, H. E., Ou,H. H., Ho,

Y.T., and Lu, T. M. 1978. In “Proceedings of Symposium on Plant Tissue Culture,” pp.

11-22, Science Press, Peking.

Chen, L., Xu, Z., Yin, G.C. Zhu, Z. Y., and Bi, F. Y. 1979a. Acra Genet. Sin. 6 , 421-426.

Chen, Z. H., Qian, C. F., Qin, M., W a g , C. H.,SUO,C. J . , Xiao, Y.L., and Hsu, H. 1979b. In

“Annual Report of the Institute of Genetics, Academia Sinica,” pp. 88-90.

Chen, S. L., Tian, W. Z . , and Zhang, G.H. 1980a. In “Annual Report of the Institute of Genetics,

Academia Sinica,” pp. 100-102.

Chen, Y., Wang, R. F., Tian, W. Z., Zuo, Q. X., Zheng, S. W. Lu, D. Y., and Zhang, G.H.

1980b. Acta Genet. Sin. 7. 46-54.



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