Plant Science Lettem, 23 (1981) 139--145 139 Elsevier/North-Holland Scientific Publishers Ltd.

IMPROVED ANTHER CULTURE OF MAIZE (ZEA MA YS)

Y.C. TING, MARGARET YU and WAN-ZHEN ZHENG

Department of Biology, Boston College, Chestnut Hill, Boston, MA 02167 (U.S.A.)

(Received April 23rd, 1981) (Revision received June 17th, 1981) (Accepted June 19th, 1981)

SUMMARY

Culture of anthers of maize hybrids Dan-San 91, King Hwang 13 and Coe's inbred stock 6 in vitro was attampted. Dan-San 91 repeatedly per- formed efficiently; approx. 17% of the inoculated anthers of this hybrid on the average responded positively to culturing. It is now possible using this hybrid to produce enough calli and embryoids within a reasonable length of time for use in basic and applied research.

Six different callus lines have been selected and these have been subcul- tured for more than 6 months. These callus lines vary in chloroplast content, growth rate and frequency of regeneration.

INTRODUCTION

Research concerned with obtaining cultures of anthers from maize (Zea mays L.) has been in progress for a decade. It was thought that maize anther cultures would be useful for the production of plants from pollen and also for studies of cytogenetics, developmental and molecular biology. However, compared with the development and successful application of anther cul- tures of Oryza and Nicotiana, the use of maize has been limited mainly by the unavailability of an efficient method.

In 1975, maize 'pollen-plants' were obtained through anther culture in vitro [ 1 ]. Since then, notable advances have been made in the area of maize anther culture with an emphasis largely on nutritional requirements, regener- ation response and selection of desired genotypes [2--4]. To produce a large number of pollen-plants and calli remains a difficult task, since most of the previous work reported that the frequency of callus and embryoid initiation

Abbreviations: 6-BA, 6-benzyladcnine; 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, naphthalene acetic acid.

0304--4211/81/0000--0000/$02.50 © Elsevier/North-Honand Scientific Publishers Ltd.

140

was on the average less than 1% of the total numbers of anthem cultured. The regeneration frequency of the calli was no more than 10%. The low frequency of regeneration and callus growth makes meaningful experiments prohibitive with respect to cost and time. Even though some reports [3,4] stated that more than 10% regeneration of calli into pollen-plants had been obtained, it was frequently difficult to reproduce the results (Ting, unpu~ fished). The present report describes our most recent researches directed towards developing a refined and reliable procedure for maize anther culture including methods for the selection of genotype.

MATERIALS AND METHODS

Materials. The maize hybrid Dan-San 91, from a cross between an inbred Pa-Tang-Be strain and the hybrid FI of inbred Shao-Pa-Tang and inbred Shui- Be lines, was used in the present work. The crosses were made in Jilin pro- vince, north-eastern China. These inbred lines have adapted to the climate of north-eastern China for a long time. The kernels of Dan-San 91 were kindly provided by Ms. Qui-hua Mu of the Jilin Institute of Agriculture, Jilin, China. Before this research was started, anthers from Dan-San 91 were tested in culture by Ms. Mu and Mr. Yang and their preliminary results indicated that this hybrid was promising [5].

The other maize mateial employed was King Hwang 13. It is the FI hybrid between inbred Pa-Tang-Be and inbred Chiao-Dan -Yihao plants. This inbred Chiao-Dan-Yihao came from the northern part of China. The hybrid kernels were a gift of the Institute of Genetics, Academia Sinica, Beijing, China. Gu et al. [3] reported that anthers of King Hwang 13 responded favorably to culture by producing more than 1% callus and embryoid from a total of more than 600 anthers inoculated.

The third maize strain utilized for this research was named Coe's stock 6 which is an inbred line. This was originally obtained from Dr. E.H. Coe of the University of Missouri in 1965. Since then it has been maintained here by selfing yearly.

Methods. The culture medium adopted for this study was named Zheng-14 [5]. One liter contains: KNO3, 3000 mg; (NH4)2SO4, 150 mg;MgSO4.7H20, 450 mg; CaC12.2H20, 150 mg; KH2PO4, 600 mg; MnSO4.H20, 10 mg; ZnSO4.7H20, 2 mg; H3BO3, 3 mg; KI, 0.75 mg; CuSO4.5H20, 0.0025 mg; CoC12.6H20, 0.0025 mg; NaMoO4.2H20, 0.25 mg; glycine, 2 mg; thiamine/ HC1, 10 mg; pyridoxine hydrochloride, 1 mg; nicotinic acid, 1 mg; Na2EDTA, 37.3 mg; FeSO4.7H~O, 27.8 mg; 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg; 6-benzyladenine (6-BA), 1 rag; a-naphthalene acetic acid (NAA), 1 mg; inositol, 100 mg; lactalbumin hydrolysate, 500 mg; sucrose, 15%; activated charcoal, 0.5%; agar, 0.6--0.8%. Before adding the agar, the pH of the medium was adjusted to 5.8. This medium with sucrose reduced to 3% was used in regeneration experiments.

The prepared medium was autoclaved at 20 lb/in 2 for 20 min. Aliquots

141

of the medium (25 ml) were dispensed into sterile Petri dishes. Maize influor- escences were collected from vigorous field-grown plants in the summer of 1980 in the Boston area. Anthers with microspores at the mononucleate stage were selected by actual observation under an ordinary light micro- scope after the spores had been stained with KI solution (1% (w/v) in 95% ethanol). Sterilization of the anthers was carried ou t under aseptic condi- tions by immersing them in 2% (w/v) sodium hypochlori te for 8--10 min, fol lowed by transfer to 70% ethanol for 5 min. The anthers were washed 3 times with sterile distilled water.

Inoculations of the anthers were done under an Edgar hood, which is an aseptic chamber. On the average, from 30--50 anthers were implanted in the medium contained in a Petri dish. Temperature in the culture room ranged from 25°C to 28°C and humidi ty varied 50--55%. After 6 weeks in total darkness in an environmental growth chamber (M-32), the cultures were illuminated daily for 16 h with cool white f luorescent lamps at an intensity of 2300--2500 lux. These growth conditions were previously reported in Ting et al. [6] .

RESULTS AND DISCUSSION

For Expt. 1, only Zheng-14 medium was used. Maize anthers of both Dan-San 91 and King Hwang 13 plants with microspores at the mononucle- ate stage were excised and inoculated into the medium. It was observed that 4--6 weeks after implantation, on the average, the initiation of growth became apparent on the surface of some anthers. The site of these initiations of growth varied from anther to anther. In the first week after growth initia- tion, it was not apparent whether calli or embryoids had formed; what was present appeared to be a mass of cells protruding out of the anther walls. In the second week after growth initiation, the morphological differences between calli and embryoids gradually became obvious. The embryoids were shining and smooth on the surface of the cell mass (Fig. 1, small arrow) while the calli were generally dull and rough (Fig. 1, large arrow). In addition, the embryoids grew faster than the calli. Two weeks after growth initiation, the embryoids became irregular in shape indicating differences in the rate of development in various parts of cell mass. Subsequently, a green area due to the occurrence of chloroplasts appears. This was consistently fol lowed by the emergence of a new shoot (Fig. 2). In most cases, the shoots were initi- ated earlier than the main roots of the embryoids. If the main roots emerged before the shoots, this was always followed by the development of profuse root systems and these embryoids showed incomplete morphogenesis, i.e. the to t ipotency of the embryoids was not realized.

CaUi remained undifferentiated for 2 weeks after growth initiation. They appeared as a mass of cells with a rough and spinule-like surface. Usually, in the third week after growth initiation some of the calli became brown and solid in appearance while the others were still creamy-white and friable. The

142

Fig. 1. Anthers of maize Dan-San 91 were cultured on Zheng-14 medium for 6 weeks. All of them show a certain amount of growth, either into callus (large arrow) or into embry- oids (small arrow) (× 2).

brown calli gradually degenerated and died about 2 weeks later. The creamy- white caUi, on the contrary, continued to proliferate abundantly. Many of them gave rise to plantlets when later transferred to regeneration medium.

For Dan-San 91, as shown in Table I, there were 1960 anthers implanted. Among them, 80 anthers, or 4.08%, grew into callus, and 266 anthers, or 13.5%, grew into embryoids. In contrast, for King Hwang 13, it was observed that among 4028 anthers cultured, only 18 of them, or 0.44%, grew into callus, and 64 of them, or 1.6%, grew into embryoids. It is clear that Dan-San 91 was invariably better than King Hwang 13 in terms of the pro- duction of calli and embryoids under uniform growth conditions.

In order to test the validity of Expt. 1, it was necessary to repeat it. The materials employed for Expt. 2 were (in addition to maize hybrids Dan-Dan 91 and King Hwang 13) an inbred maize, Coe's stock 6 (Stock 6 for short). The medium was the same as for Expt. 1. The results obtained are also listed in Table I. In this table, it is clearly seen that for Dan-Dan 91, 3920 anthers were cultured in vitro, 641 of them, or 16.3%, responded positively by growing into calli and embryoids. However, it is regrettable that due to an accident, the separate records of the number of calli observed and that of

143

Fig. 2. Embryoids from anthers of maize Dan-San 91 after 9 weeks of culture on medium Zheng-14. The arrow points to a newly-regenerated shoot (x 4.5).

embryoids were unavailable. Hence, the record of Expt. 2 is not as informa- tive as that of Expt. 1. For King Hwang 13, 611 anthers were implanted, 31 of them, or 5.0%, grew into calli and embryoids. In contrast, only 215 anthers of Stock 6 were available for culturing, two of this small sample of

TABLE I

NUMBER AND PERCENTAGE OF MAIZE ANTHERS FROM HYBRIDS DAN-SAN 91, KING HWANG 13 AND STOCK 6 THAT GREW INTO CALLI AND EMBRYOIDS ON MEDIUM ZHENG-14

Genotype No. anthers Calli Embryoids Calli and embryoids inoculated initiated obtained obtained No. (%)

No. (%) No. (%)

Dan-San 91 Expt. 1 1960 80 (4.1) 266 (13.5) 346 (17.6) E x p t . 2 3 9 2 0 - - -- 641 (16.3)

King Hwang 13 Expt. 1 4028 18 (0.4) 64 (1.6) 82 (2.0) E x p t . 2 6 1 1 - - -- 31 (5.0)

Stock 6 215 -- - - 2 (I .0)

144

215 anthers, or 1%, did the same. This is markedly below the frequency of occurrence of calli and embryoids for either Dan-San 91 or King Hwang 13 as described in the foregoing experiment.

When the two experiments are compared, it is interesting to note that the results for Dan-San 91 and King Hwang 13 of Expt. 1 are very close to those of Expt. 2. For instance, in Table I, if the number of calli and that of embryoids are considered together, the similarity in results between the two experiments becomes very convincing. For Dan-San 91, the sum of the numbers of both calli and embryoids obtained will be 346 (80 + 266), or 17.6% of the total. This percentage is very close to 16.3% which resulted in Expt. 2 for the same hybrid. Furthermore, for King Hwang 13, if the number of calli and that of the embryoids are added together, the sum will be 82 (18 + 64), or 2%, which is probably significantly different from the

Fig. 3. F o r m a t i o n of shoots , roots , as well as leaves, 4 weeks after the calli f rom the anthers o f maize Dan-San 91 had been o n the regenerat ion m e d i u m . (Original size).

145

5% found in Expt. 2 for the same hybrid. This probably can be accounted for by the difference in sample size between the two experiments.

When the calli of King Hwang 13 were 4--6 weeks old, some of them were transferred to regeneration medium for testing. This medium is similar to Zheng-14 used for calli initiations in terms of the amounts of major elements, hormones and growth regulators except that sucrose was reduced from 15% to 3%. Light intensity, temperature and length of daily illumina- tion etc. were kept the same as for calli initiations. After the calli has been on the regeneration medium for 1--2 weeks, embryogenesis began as evi- denced by the growing out of yellow-green shoots. This was followed by the differentiation of roots. When morphogenesis occurred in this order, shoots before roots, the result was usually healthy plantlets. On the contrary, if roots were formed before shoots, degenerated embryos were usually formed. From our preliminary tests, it was estimated that more than 50% of the calli under these conditions would undergo regeneration. The plantlets were generally healthy and vigorous (Fig. 3). Since 10 chromosomes were present in most of the root-tip cells, these plantlets were certainly pollen derived plants (n = 10).

CONCLUSION

The results of our experiments indicate that the efficiency of maize anther culture has been improved. With hybrid Dan-San 91, 16.3--17.6% of the plated anthers grew into callus and embryoids. These percentages are greater than those in all previous reports. With Dan-San 91 and medium Zheng-14, it is now possible to produce enough calli and poUen-plants within a reasonable length of time for studies of genetics, breeding and developmental biology.

REFERENCES

1 Research Group No. 4, Genetics Institute Academia Sinca, Beijing, China, Acta Cenet. Sinica, 2 (1975) 138.

2 Z. Opatrny, J. Dostal and V. Martinek, Bio. Plant. (Praha), 19 (1977) 477. 3 M.K. Gu, W.C. Cheng, L.C. Kuo, Y.L. Kwan, H.P. An and C.H. Hwang, in: Pro-

ceedings of Symposium of Plant Tissue Culture, Science Press, Peking, 1978, p. 35. 4 Z.S. Guo, A.C. Sun, Y.Y. Wang, Y.L. Gui and S.H. Miao, Acta Genet. Sinica, 20

(1978) 204. 5 A.H. Mu and Z.T. Yang, Acta Phytophysiol. Sinica, 5 (1979) 291. 6 Y.C. Ting, A.M. Boyer and G. McSweeney, Maize Genet. Coop. Newslett., 52 (1978)

6.