Cell-Suspension Culture of Maize (Zea mays L.)

Institute of Plant Physiology and Biochemistry, Siberian Branch, USSR Academy of Sciences, Irkutsk, USSR

Cell-Suspension Culture of Maize (Zea mays 1.) ROSA T. POLIKARPOCHKINA, K. Z. GAMBURG and E. E. KHAVKIN With 2 figures Received March 14, 1979 . Accepted May 14, 1979

Summary A shake flask culture was established from maize (hybrid C 103 X W 155) callus initiated at nodal and internodal stem segments and maintained in the medium containing the inorganic constituents of MURASHIGE and SKOOG (1962), 20 gil sucrose, 0.4 mgll thiamine, 1 gil casein hydrolysate and 1 mgll 2,4-D. Small aggregates that lacked inner organization were predominant in the suspension. Cells were of a parenchyma-type, mostly round-shaped, and their sizes were distributed in log-normal pattern (the mean diameter 40-50 ft). The exponential growth in the batch-grown culture lasted to 4th-5th day, the transition to the stationary phase took place during the 5th-6th day, and after the 9th day a considerable degradation was observed in the culture. Doubling periods for the cell number, and dry matter and protein during the exponential growth were about 28 h at 26 DC, and the total increase was at least IS-fold during the 7-day subculture cycle. Respiration rate on the fresh weight, protein and cell basis was constant from the 1st to the 6th day and then declined 2-fold, however, it was completely restored during the first 12 h after cell transfer to the fresh medium. Key words: batch suspension culture, cell-size distribution, nutrient requirements, protein, respiration, Zea mays.

Introduction

Suspension cultures provide unique possibilities for cytogenetic studies of somatic cells and for clonal selection of characteristic epigenotypic patterns; they facilitate the detailed investigation of nutritional requirements of plant cells and the regulatory mechanisms of their metabolism, and also - with unavoidable reservations - open an entirely fresh approach to the studies of cell growth and differentiation (for review see BUTENKO, 1978; KING and STREET, 1977; STREET, 1977). Cereals are especially attractive because of their agricultural importance, however, the establishment of their isolated cell cultures presents considerable technical difficulties (YAMADA, 1977). Maize occupies an outstanding place among cereals as its genetics and biochemistry have been extensively studied and vast collections of genetically defined strains are available as a starting material for isolated cultures. However, Z. PJlanzenphysiol. Bd. 95. S. 57-67. 1979.

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ROSA T. POLIKARPOCHKINA, K. Z. GAMBURG and E. E. KHAVKIN

most of maize somatic cell cultures derived from developing and mature embryos and propagated in several laboratories (see KING et al., 1978, for detailed bibliography, and also BUTENKO et al., 1978; KHAVKIN et al., 1978, 1979) proved to be pseudo-callus cultures interpreted either as aberrant root-like clusters (MoTT and CURE, 1978) or as tissue-culture homologs of root terminal and lateral meristems (FREELING et al., 1976). Recently the establishment of somatic cell-suspension cultures of maize has been repeatedly reported. However, the culture described by OSWALD et al. (1977) consists of low-viable cells that survive after separation from large pseudo-callus clumps. GREEN (1977) also reported an embryo-derived callus maintained in suspension culture. POTRYKUS et al. (1977) established a rapidly growing callus culture from protoplasts originating from internodal cells of 8-week-old seedlings, a selected cell line was maintained in liquid culture as small cell aggregates. The authors (KING et al., 1978) regard it a finely dispersed suspension. Just lately a rapidly growing suspension culture was also communicated by SANCHES DE JIMENEZ et al. (1978). However, in the publications available growth parameters of the cultures were not described in sufficient detail for a comparison of maize suspension with the well-defined suspension cultures of dicots. In this paper we report the establishment and growth parameters of batch-grown cell-suspension culture apparently similar to that described by KING et al. (1978). In contrast to the latter, we obtained callus directly from stem explants and then selected friable rapidly growing portions of callus for further propagation and initiation of suspension culture. The rapidly growing suspension culture described here consists of small aggregates that lack noticeable inner organization and apparently presents a satisfactory and highly reproducible experimental system for further physiological and biochemical studies of maize cells. Some of these data have already been published in short elsewhere (KHAVKIN et al., 1979; POLIKARPOCHKINA et al., 1979). Material and Methods Source, establishment and maintenance 0/ callus and suspension cultures Seeds of hybrid C 103 X W 155 were obtained from the Krasnodar Agricultural Research Institute. Plants were grown for 30-40 days in a greenhouse or in a phytotron chamber (22,000 lux, 16 h day at 30 ° and 20°C at night). 2 to 3-cm-long sections including nodes were excised from the 4th and the 5th internodes, surface-sterilized for 15 min in a 0.15 Ofo Diocidum (ethanol mercuric chloride plus N-cetylpyridinium chloride, 2: 1) solution and rinsed copiously with sterile distilled water. 2-mm-thick cross-sections were cut in the intercalary meristem region and just above it and placed 2-3 per 125 ml Ehrlenmeyer flask onto 40 ml nutrient medium containing the mineral salts of MURASHIGE and S~OOG (1962), 20 gil sucrose, 1 gil pancreatic casein hydrolysate (CH), 80 mgll inositol, 0.4 mg/l thiamineNOs, 0.1 mg/l pyridoxine-HCI, 15 mg/l 2,4-D and 8 gil Bactoagar. Explants were incubated in the dark at 33°, and after 15-20 days several small calli usually appeared on the perifery of the sections and later fused into a light-coloured firm and rapidly growing callus. Z. P/lanzenphysiol. Bd. 95. S. 57-67. 1979.

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During subsequent callus propagation 2,4-D concentration was lowered to 6 mg/l. Every 30 days ca. 150 mg inocula per flask selected from most friable parts of callus were transferred to a fresh solid medium, and the fresh weight of callus increased ca. 50-fold by the end of the subculture. After 5 such passages ca. 500 mg stock callus inocula were transferred to 250 ml Ehrlenmeyer flasks and dispersed in 60 ml of liquid medium of the same composition as described above, except that agar was omitted and 2,4-D concentration lowered to 2 mg/l. Suspension was grown on a reciprocal shaker at 80 strokes per min at 26°C in the darkness. Five-six days later friable aggregates not exceeding 2 mm in size became predominant in the suspension and its density increased rapidly. The suspension was propagated by subculturing at first every 10 days (16 successive passages) and then every week by a 20-fold dilution (0.6 to 1 g inoculum was pipetted to 60 ml of fresh medium). In some experiments designed to study nutrient requirements 5 ml samples of suspension were grown in 16 X 150 mm test tubes in a roller at 60 r.p.m. Analytical methods Tissue samples were harvested by suction through a glass filter on the designated days in the course of the 12th to 53rd subcultures, fresh and dry weight was determined and the tissue was sampled for cell and mitotic index counts and for determination of protein nitrogen and respiration. Cell numbers were counted in a haemocytometer slide after maceration in 10 % (w/v) chromic trioxide; cell size (area) and shape were estimated in ca. 200-cell populations by drawing the cells and then measuring two perpendicular axes and weighing the cell images. To estimate the mitotic index, samples were fixed in ethanol: acetic acid: formalin (18 : 6 : 1) for 2 h, kept in 70 % ethanol, stained with Feulgen after 30 min hydrolysis in 6 N HCI at room temperature, post-stained in a Light green and embedded in saturated sucrose solution in glycerine. The drops of stained suspension were used for preparing of slide preparations immediately before counting. At least 3,000 cells were investigated in each sample, and the mitotic index was calculated as a sum of metaphase, anaphase and telophase figures per 1,000 cells. Protein nitrogen was determined by a micro Kjeldahl procedure after precipitation of proteins with 5 Ofo (w/v) trichloroacetic acid or 70 Ofo ethanol. Oxygen consumption was assayed polarographically with a Clark probe attached to LP-7 polarograph and EZ-7 linear recorder in a 2-ml Lucite cell at 26°C. Two media were employed in these experiments: a hypertonic medium (660 mM mannitol, 20 mM KH2P0 4, 10 mM KCI, 5 mM MgCI 2 , pH 5.8) and an isotonic pH 5.8 medium used for suspension culture. The initial O 2 concentration was 240 mM, and the recorder was calibrated using yeast suspension. The relative analytical error did not usually exceed 5 Ofo. All experiments were run at least in triplicate.

Results Nutrient requirements for batch suspension culture

A series of tests demonstrated that omission of 2,4-D from the medium completely blocked growth in the first subculture and caused cell death. Under optimal 2,4-D concentrations (0.5 to 2 mg/I) high growth potential was maintained in subsequent subcultures, while suboptimal 2,4-D level (0.2 mg/I) did not immediately influence the growth rate, however, its subsequent growth was diminished and the culture died notwithstanding the permanent presence of low 2,4-D in the medium (Figure 1,

z.

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60

ROSA

r. POLIKARPOCHKINA, K. Z. GAMBURG and E. E. KHAVKIN c

B

A

-

120

~ ~

r-

60

l"""~

~

N

0

o

0

It)

0

-

N

r:-

It)

0

0

d

N

0

It)

0

-

N

Gl

Fig. 1: The effect of nutrient concentration on growth of the maize suspension culture (fresh weight by the end of 7-day batch cycle, mg/ml suspension. A, sucrose, gil; B, casein hydrolysate, g/I; C, 2,4-D, mg/1. Table 1: Maintenance of growth of the maize suspension culture as related to several constituents of nutrient medium. Medium composition

Final fresh weight after 7-day-Iong culture in successive passages, mg / ml 1st 2nd 3rd

Complete;') Same minus thiamine Same minus inositol Same minus pyridoxine Same minus casein hydrolysate Complete except 2,4-D 0.2 mg/I Complete except 2,4-D 0.5 mg/I

125 110 131 120 66 100 111

117 22 104 122 52 57 109

119

';"f)

119 117 68 *,;.) 110

'f) thiamine-HNO a 0.4 mg/I; inositol 80 mg/I; pyridoxine-HCI 0.1 mg/I; casem hydrolysate 1 g/I; 2,4-D 2 mg/1. ,;.,;.) cells died.

Table 1). Thus optimal auxin concentration is necessary to maintain maize suspension culture, and its growth rate cannot be regulated by 2,4-D level continuously. The omission of thiamine led to drastic growth inhibition in the 2nd passage and cell death in the 3rd passage. The omission of inositol and pyridoxine produced no Z. Pjlanzenphysiol. Bd. 95. S. 57-67. 1979.

Maize cell suspension cultures

61

adverse effect on culture growth and maintenance (see Table 1). The omission of CH twice reduced the growth rate in the 1st and subsequent passages, however, cell death did not occur in this case. Preliminary experiments demonstrated that CH could be replaced by 5-10 mM asparagine or Na-glutamate. Therefore the following standard medium was used throughout all the subsequent experiments: the MURASHIGE and SKOOG salts, 20 gil sucrose, 0.4 mgll thiamine-NO a, 1 gil CH and 1 mg/12,4-D. Cell parameters of batch suspension culture As most of plant suspension cultures, maize suspension consisted of aggregates of various sizes, mostly 0,1 to 1 mm. Serial filtration of suspension through nylon sieves (Table 2) showed that aggregates 0.2 to 0.8 mm and > 0.8 mm predominated by the end of the exponential phase, while the latter fraction accumulated significantly during next three days. Microscopic observations revealed that large aggregates consisted of loosely connected small ones devoid of characteristic meristem-like zones and inner organization (Figure 2). Single cells comprised less than 0.1 Ofo total cell number. When a small (up to 10 cells) aggregate fraction was used as inoculum, soon large friable aggregates reappeared and the initial level of aggregation was restored by the end of the passage. All cells were of a parenchyma-type and revealed numerous cytoplasmic strands and amyloplasts especially on the 2nd-3rd day of the subculture cycle. No xylem elements were found in macerates. Though a small number of cells were elongated, most cells were nearly round-shaped: in ca. 75 Ofo cells the long to short axis ratio was not over 1.5, and this shape distribution was similar in the exponential and stationary phases (Table 3). Cells varied widely in size. Area distribution was lognormal and in the stationary phase the median shifted towards larger cells proper to a 1.6-fold increase in the cell volume (Table 4). Cell size variation was independent of shape distribution. Table 2: Size distribution of aggregates experiments).

In

the maize suspension culture (mean of two

Fractions

Per cent of total fresh weight 4th 7th days of culture

<0.2mm 0.2-0.8 mm 0.8mm

15 44 41

>

8 23

69

Growth of batch culture The growth of the suspension was monitored by the mitotic index, cell number, fresh and dry weight, and protein content (Table 5). When the initial suspension

z.

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62

ROSA

T.

POLIKARPOCHKINA,

K. Z.

GAMBURG

and E. E.

KHAVKIN

Fig. 2: Cell aggregation in 7-day-old maize suspension culture. A, large aggregates; B, small aggregates.

density was 10 mg/ml (ca. 105 cells) cells multiplied and accumulated dry matter and protein lS-18-fold during the 5 days of the subculture cycle. These parameters did not change further from the 5th to 8th day and decreased sharply by the 9th day presumably due to degradation of the culture. As the fresh weight accumulation lagged behind that of dry matter and behind cell proliferation, cell counts, and dry matter and protein content on fresh weight basis were temporarily raised in the exponential phase. This change of dry matter content could be attributed both to Z. Pjlanzenphysiol. Bd. 95. S. 57-67. 1979.

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Table 3: The variability of cell shape in the maize suspension culture. The long to short axis ratio, classes

1.00-1.10 1.11-1.20 1.21-1.30 1.31-1.40 1.41-1.50 1.51-1.60 1.61-1.70 1.71-1.80 1.81-1.90 1.91-2.00 2.01-2.50 2.51-3.00 total number of cells measured

Frequencies, 0/0 the 1st experiment the 2nd experiment 2-day-Iong 7-day-Iong 2-day-Iong 7-day-Iong culture culture culture culture 14.3 17.1 19.9 16.2 6.0 9.3 5.2 4.4 1.8 1.8 4.0 1.0 226

9.8 19.1 16.0 10.2 8.9 9.3 5.8 3.1 4.9 3.6 4.4 3.0 225

14.0 21.0 20.6 14.4 11.7 1.2 3.5 5.0 1.2 3.1 3.5 0.4 257

14.4 21.0 20.0 15.3 12.4 6.2 2.9 2.4 1.0 1.0 2.0 1.0 209

Table 4: The variability of cell sizes in the maize suspension culture (the number of measured cells same as in Table 3). Lg cell image area (.u 2), classes

Frequencies, 0/0 the 1st experiment the 2nd experiment 2-day-long 7-day-long 2-day-Iong 7-day-long culture culture culture culture

2.40-2.49 2.50-2.59 2.60-2.69 2.70-2.79 2.80-2.89 2.90-2.99 3.00-3.09 3.10-3.19 3.20-3.29 3.30-3.39 3.40-3.49 3.50-3.59 3.60-3.69 3.70-3.79 3.80-3.89 3.90-3.99 4.00-4.09 4.10-4.39

1.8 1.3 4.0 4.4 6.2 9.3 11.0 17.2 11.5 9.3 10.6 5.3 0.9 2.6 1.8 1.8 1.3 0

0 0 1.8 1.3 7.1 6.2 10.2 11.6 12.0 7.6 11.1 9.8 6.7 5.3 3.1 2.7 1.8 1.8

0.8 1.2 1.2 3.9 6.2 10.1 12.5 11.7 14.4 18.3 9.3 3.1 2.3 2.3 1.9 0.8 0 0

the median mean area (f.-t2) mean volume (f.-t3)

3.208 1,620 64,000

3.326 2,120 97,300

3.198 1,580 60,700

0 0 1.0 2.4 3.3 5.7 11.0 14.8 10.5 12.0 12.4 5.3 6.7 6.2 3.3 2.4 1.4 1.4 3.337 2,170 101,300

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ROSA T. POLIKARPOCHKINA, K. Z. GAMBURG and E. E. KHAVKIN

absorption of low molecular substances and to accumulation of 70 Ofo-ethanol-insoluble components. The fresh weight increase during the 6th to 8th day was apparently due to water uptake. Cell size values calculated from the area measurements agreed well with the volume data obtained by cell counting in macerates (compare Tables 4 and S). For example, mean 10 6 cell volumes were 62 and 99 mm 3 in exponential and stationary phases, while mean weights of 106 cells were 70 and 120 mg, respectively. Table 5: Growth parameters of the maize suspension culture (mean of at least 3 experiments). Days of the subculturing cycle

Cell number, Mitotic X 10-6 index per per g mlof fresh susweight pens iIon

0 2 4 5 6 7 8 9

3 38 24 21

s.e./mean

17

0.086 7.7 0.28 12.8 0.70 11.5 1.56 12.8 1.27 9.2 8.5 1.33 1.51 8.5 1.20 7.4

6 4 5

20

Fresh weight, mg per per mlof 106 sus- cells pension

Dry weight, mg per per g per mlof fresh 10 6 susweight cells pension

Protein nitrogen, mg per per g per mlof fresh 106 susweight cells penSlon

11 22 61 122 138 156 177 163

0.67 64 2.24 102 6.47 106 9.88 81 10.21 74 10.14 65 11.15 63 8.80 54

0.027 0.091 0.24 0.45 0.40 0.40 0.41 0.32

130 78 87 78 109 117 117 136

15

8.3 7.9 9.2 6.3 8.1 7.6 7.4 7.3

6

2.45 4.12 3.97 3.72 2.90 2.57 2.32 1.96

0.32 0.32 0.35 0.29 0.31 0.30 0.27 0.27

5

0/0

As cell proliferation and dry matter accumulation during the first two days were slower than in the following two days, a small lag-period was presumed to exist in the culture cycle. Let us assume that the subsequent relative growth rate is constant through 4 days of subculture. Then the lag-period (tl) and doubling time (td) are calculated as tl

Ig P 2 -Ig Po )

= 48 ( 1- Ig P 4 -Ig P 2

and td

Ig 2

= 48 Ig P IP 4- g 2

where Po, P 2 and P4 are parameter values on the zero, 2nd and 4th day of the subculture. The data calculated in this way are presented in Table. 6. Thus under conditions described above the subculture cycle consists of 6 to 12 hlong lag-period, 90 to 110 h exponential growth phase with the cell number and dry mater content doubled appoximately four times, a short transitional period (the Sth to the 6th day), stationary phase (the 6th to the 8th day) and a subsequent period of degradation. Z. P/lanzenphysiol. Bd. 95. S. 57-67. 1979.

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Table 6: The lag-period and doubling time in the maize suspension culture. Parameters (per ml)

Period, h lag (tl)

The number of doubling (td) doublings per subculture

cell number dry matter protein nitrogen

11 6 9

28.2 27.5 27.4

4.2 4.0 4.1

Respiration Two kinds of experiments were designed for determination of respiration rates. A sample of filtered tissue was put into the cell and fresh mannitol or sucrose medium was poured over it, then after the Clark probe was inserted, the mixture was stirred. In alternative experiments tissue samples were suspended in fresh culture medium, preincubated for 1 h at 26 DC with occasional agitation and the polarographic cell was filled with this suspension. The rate of O 2 consumption was not influenced by the incubation medium, and preincubation in fresh medium did not produce any respiration enhancement either in exponential or in stationary phase cells (Table 7). Endogenous respiration substrates seem therefore to be used exclusively during the whole culture cycle. Table 7: The effect of incubation medium and preincubation on respiration rates of the maize suspension culture. Days of subculture cycle

Respiration, ,umole 02/min without preincubation mannitol nutrient medium medium

per g fresh weight I-h-preincubation on nutrient medium

3 4 7

1.82 2.05 0.87

1.75 2.20 0.90

1.93 0.89

Table 8: Respiration rates in the batch cycle of the maize suspension culture, ,umole 02/min (mean of 6 experiments).

per g fresh weight per mg protein nitrogen per 108 cells

Days of the subculture cycle 0 1 2 3 4

5

6

7

0.6

1.5

1.5

1.4

0.7

0.47

0.28

1.5

1.8

1.5

0.24

0.42

0.38

0.40

0.08

0.14

0.13

0.11

0.15

0.08

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ROSA T. POLIKARPOCHKINA, K. Z. GAMBURG and E. E. KHAVKIN

Compared to the stationary phase, respiration doubled during initial 24 h after transfer to fresh medium. Additional experiments proved that significant enhancement of respiration occured at least 2-4 h after the transfer to the fresh medium and by 10 to 12 h O2 consumption reached the high level which was maintained up to the 6th day and was followed with abrupt decline on the 7th day (Table 8). Discussion By now maize cell-suspension culture has been maintained in this laboratory by serial culture through more than 70 transfers (mostly in 7-day cycles), and the reported growth and metabolic parameters appeared to be highly reproducible. Yet the factors that determine the initiation of friable callus capable to grow in suspension are unknown. Such calli were repeatedly obtained in the case of C 103 X W 155 strain and also in one experiment with stem explants of inbred W 155 (in the latter case a suspension culture was established but later lost). Similar suspension culture was established from stem cells of inbred 2717 (KING et aI., 1978), and two maintained suspensions seem to differ mostly in their degree of aggregation that can be genotype-dependent. However, no suspension cultures have been obtained for the present from other investigated maize strains in our laboratory. Growth and metabolic parameters of maize culture reported here are comparable to those of most rapidly growing batch cultures of tobacco, bush bean, rose, soybean, rice, maize and Datura cells (see review by NOGUCHI et aI., 1977, and also KING et aI., 1978; VYSOTSKAYA and GAM BURG, 1975) that have td of 20 to 30 h. Characteristic features of our maize suspension are a rather short lag-period and high dry matter content (up to 10 % in exponentially growing culture) comparable only to that of rice suspension (LIEB et aI., 1973). In contrast to a transient peak of oxygen consumption in the batch cultures of tobacco, sycamore and rose suspensions (BELLAMY and BIELESKI, 1966; GIVAN and COLLIN, 1967; NASH and DAVIES, 1972), maize cell respiration did not decline after a rapid increase at the onset of proliferation and was preserved on a high level throughout the whole exponential phase. Acknowledgements We are thankful to Professor R. G. BUTENKO for helpful advice and to L. M. OSHAROVA, T. P. POBEZHIMOVA and O. V. PYSARSKAIA for expert technical assistance.

References BELLAMY, A. H. and R. L. BIELESKI: Austr. J. BioI. Sci., 19,23-36 (1966). BUTENKO, D. G.: FisioI. Rast. (Moscow), 25, 1009-1024 (1978). BUTENKO, R. G., L. V. KUZNETSOV, O. V. SKRIPKA, and E. A. ZJATKOVA: FisioI. Rast. (Moscow), 25, 166-170 (1978). FREELING, M., J. C. WOODMAN, and D. S. K. CHENG: Maydica, 21, 97-112 (1976).

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GIVAN, C. V. and H. A. COLLIN: J. Exptl. Bot., 18, 321-331 (1967). GREEN, C. E.: Hortscience, 12, 131-134 (1977). KHAVKIN, E. E., S. I. MISHARIN, L. E. MONASTYREVA, R. T. POLIKARI\OCHKINA, and T. B. SUKHORZHEVSKAIA: Z. Pflanzenphysiol., 86, 273-277 (1978). KHAVKIN, E. E., S. I. MISHARIN, and R. T. POLIKARPOCHKINA: Planta (Berl.), 145,245-251 (1979). KING, P. J., I. POTRYK.US, and E. THOMAS: Physiol. veg., 16, 381-399 (1978). KING, P. J. and H. E. STREET: In: STREET, H. E. (Ed.): Plant Tissue and Cell Culture (Botanic Monographs, vol. 11), 307-387. California Univ. Press, Berkeley and Los Angeles, 1977. LIEB, H. B., T. B. RAY, and C. C. STILL: Plant Physiol., 51,1140-1141 (1973). MOTT, R. L. and W. W. CURE: Physiol. Plant., 42,139-145 (1978). MURASHIGE, T. and F. SKOOG: Physiol. Plant., 15,473-497 (1962). NAsH, D. T. and M. E. DAVIES: J. Exptl. Bot., 23, 75-91 (1972). NOGUCHI, M., T. MATSUMOTO, Y. HIRATA, K. YAMAMOTO, A. KATSUYAMA, A. KATO, S. AZECHI, and K. KATO: In: BARz, W., E. REINHARD and M. H. ZENK (Eds.): Plant Tissue Culture and its Bio-technological Application, 85-94. Springer-Verlag, Berlin, Heidelberg, New York, 1977. OSWALD, T. H., R. L. NICHOLSON, and L. F. BAUMAN: Physiol. Plant., 41, 45-50 (1977). POLIKARPOCHKINA, R. T., K. Z. GAM BURG, and E. E. KHAVKIN: Dokl. Akad. Nauk SSSR (Moscow), 245, 1278-1280 (1979). POTRYKUS, I., C. T. HARMS, H. LORz, and E. THOMAS: Molec. Gen. Genet., 156, 347-350 (1977). SANCHES DE JIMENEZ, E., E. FERNANDEZ, V. M. LOYOLA, and M. ALBORES: Abstr. 4th Intern. Congress Plant Tissue and Cell Culture, 177, Calgary 1978. STREET, H. E.: In: SCHUTTE, H. R. and D. GROSS (Eds.): Regulation of Developmental Processes in Plants, 192-218. Fischer Verlag, Jena, 1977. VYSOTSKAYA, E. F. and K. Z. GAMBURG: Fisiol. Rast. (Moscow), 22, 63-69 (1975). YAMADA, Y.: In: REINERT, J. and Y. P. S. BAJAJ (Eds.): Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, 144-159. Springer-Verlag, Berlin, Heidelberg, New York, 1977.

R,OSA T. POLIKARPOCHKINA, KIM Z. GAMBURG and EMIL E. KHAVKIN, Institute of Plant Physiology and Biochemistry, Siberian Branch, USSR Academy of Sciences, Irkutsk 33, P.O.Box 1243, USSR 664033.

Z. PJlanzenphysiol. Bd. 95. S. 57-67. 1979.