* Published as P a ~ r No. IOO in the Journal Series of the Experiment Station, Hawaiian Sugar Planters' Association, Honolulu, Hawaii.

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1 ROBERT E. COLEMAN 533 naturally in spring might inhibit the protrusion of flowers and cause reversion to a vegetative stage. H. F. CLEMENTS (Hawaii): The intensity of stimulus in spring may be more important in preventing subsequent bolting than the effect of long days following induction of flowering. J. N. WARHER (Hawaii): If under Hawaiian conditions we did get spring initiation, we would have ample time to observe later the signs of reversion. But this is not generally the case even in varieties which tassel heavily during the normal season. J. T. RAO (India): At Coimbatore, after keeping S. spontaneum clones in a vegetative condition by giving them four hours extra light from 4th month after planting, flowering can be induced by giving them four hours extra darkness varying from 4-6 weeks. DR. GLASZIO~: I tried the same sort of experiment with Saccharum spontaneum as Dr. Rao has described but could not induce flowering. DR. CLEMENTS : I, also, could not induce flowering in S. spontaneum using the same methods as Dr. Rao. In one experiment a very precise night length seemed to be essential to induce flowers in N:Co D. I. T. WALKER (Barbados): The possible explanation for increase in flowering after long hot water treatment may be due to the fact that owing to faster early growth there are more canes 'ripe to flower' when the critical daylength arrives. S.E.S appears sensitive to nutritional differences and quite insensitive to any precise photoperiodic control. CONTROL OF FLOWERING AND THE USE OF POLLEN STORAGE AS TECHNIQUES IN A SUGAR CANE BREEDING PROGRAMME* Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture (Presented by Dr. I. E. Stokes) A controlled breeding programme has been the objective of nearly every sugar cane growing country in the world. As a part of the United States Department of Agriculture's broad programme for sugar cane improvement, cooperative investigations on the flowering of sugar cane have been carried out for 8 years with the Experiment Station of the Hawaiian Sugar Planters' Association. The research work has followed two approaches: one towards basic factors or processes involved in flowering, and another towards the practical aspects of controlling the flowering for breeding purposes. While we are still a long way from having a complete knowledge of the intricate processes involved in the change from a vegetative to a flowering plant, we have found that certain processes are similar and fit into the scheme of flowering described by plant physiologists for piants in genera1l09l4. However, with sugar cane, the problem is complicated by the genetic complexity of the plant which results in exceptions to nearly every generalization one attempts to make. For example, we have assumed that most varieties of sugar cane are short-day plants but we do find clones that are long-day or are indeterminate in this respect. However, through our research efforts we have learned to control to some degree the flowering of certain varieties. During 1961, six thousand seedlings were produced as results of crosses in which the flowering of one or both parents had been controlled. * Published as P a ~ r No. IOO in the Journal Series of the Experiment Station, Hawaiian Sugar Planters' Association, Honolulu, Hawaii.

2 534 CANE BREEDING The information herein reported covers several aspects of this problem which may have implications in the further development of controlled breeding programmes. EFFECT OF PHOTOPERIOD It has been quite generally accepted that many varieties of sugar cane are intermediate-day plants, i.e. they flower only when the photoperiod falls within arelatively narrow range (about 12.5 hours). They could also be looked upon as forms of short-day plants since the critical photoperiod is nearly always shorter than the longest natural day, and night interruption usually prevents flowering. SACHS~~ suggested that sugar cane may be a long-short day plant. Certainly many varieties appear to be; several attempts to induce flowering on spring photoperiods were unsuccessful, supporting that suggestion. These attempts included raising mil temperatures during March, April, and May to the level of fall soil temperatures at a location where spring air temperatures are similar to fall air temperatures. The experiments were carried out with fully mature plants, both in the field and in pots. Therefore in these tests, air temperature, soil temperature, soil moisture, and the age of the plants were almost identical with those of the fall. Induction of flowering did not occur, which indicates that the plants perceive the difference of going from long days to short days as contrasted with going from short to long days. In this same connection an experiment was conducted in the fall of 1961 to determine the effect of lower than normal root temperatures on flowering. Plants of N.Co. 310 were grown with soil and root temperatures of 60" F. beginning August I. These plants initiated flowers and the flowers emerged at exactly the same time as those of control plants. Further work at this station has shown that certain varieties, such as SES 205A, are not inhibited from flowering by night interruption. A related species, Eulalia sp., initiated tassels on interrupted nights but not on natural nights and Erianthws bengalensi was unaffected by night interruption. Since the original report of successful induction with a fixed 12.5 hour photoperiod, several variations of this treatment have been investigated. showed that with the variety H , extension of the 8-hour day with 4.5 hours of supplemental light to give a 12.5-hour photoperiod can be accomplished with fluorescent or incandescent light and that the latter'with an intensity of only 3ft-ca is effective in extending the day. It was found that 4.5 hours of low-intensity supplemental light could be given as 2.25 hours before the dark period and 2.25 hours after, or as 4.5 hours before or after the dark period and still effect induction. However, tests show that induction occurred to a greater degree if the 11.5-hour dark period was immediately preceded only by sunlight and followed by 4.5 hours of low-intensity light. A treatment of 8 hours of sunlight followed by 11.5 hours of dark, then I hour of low-intensity light followed by an additional 3.5 hours of darkness resulted in no initiation which appears to indicate that this variety of sugar cane requires specifically 11.5 hours of night followed by 12.5 hours of light. It was obvious in these photoperiod experiments that when plants received only 8 hours of sunlight and the additional 4.5 hours with low-intensity light, 80 to go inductive nights were required; in nature only 15 to 20 inductive nights are required. Experiments in which the night was interrupted by light have shown that

3 ROBERT E. COLEMAN plants which receive natural day-lengths for only the period August 28 to September 12 will initiate flower $rimordia. Although corroborative experimental work is lacking, it seems probable that 8 hours of sunlight is insufficient time for the plant to produce or accumulate photosynthetic products required to produce the flowering stimuius during the dark period. It was also found that sugar cane plants would not flower when grown entirely on artificial illumination (incandescent and fluorescent) of 1000 to zoo0 ft-ca with 12.5-hour photoperiods. Additional studies of pre-induction treatments with natural days, continuous days, and 8-hour days show that they have no effect on flower induction as previously indicated by the author There was evidence from one experiment that partial induction is possible and that plants which receive a weak flowering stimulus due to fewer inductive nights will flower much later than normal or show bunch top (multiple vegetative leafing at the stem apex). In this test, plants of the variety N:Co. 310 were kept on interrupted nights beginning on August 22. On alternate days after August 28, four plants were removed from the night interruption and exposed to natural days. The results are shown in Table I. TABLE I DEGREES OF FLOWERING OBSERVED WHEN SUGAR CANE PLANTS (VARIETY N:CO. 310) WERE EXPOSED TO VARYING NUMBERS OF INDUCTIVE AND PROHIBITIVE NIGHTS DURING AUGUST AND SEPTEMBER 1960 Date plants moved from Number of natural Degree o,f fiowei in$** night interrufitzl7n* day-lengths before observed at kar oect to natural day-lengrhs or after September 12 (February I) -- August F, F, F, F August 30 I3 F, F, F, F September I I I F, F, F, F September 3 9 F, F, F, F September 5 7 F, F, F, F September 7 5 F, F, F, YT September g 3 F, F, YT, o September I I I F, F, YT, o September 13 I 0, 0, 0, 0 September 15 3 o, o, F, BT September 17 September , 0, 0, 0 F, BT, BT, BT September 21 9 o, o, o, BT September 23 I I 0, 0, 0, 0 September 25 I3 o, o, YT, BT Checlr (natural day-length) 21 8 F's Check (interrupted nights) o 14 o's, 2 YT 3 BT * Plants started on night interruption August 22, ** F = tasselled, YT = young tassel, o = no tassel, BT = bunch top. I Plants which were inhibited up to September 13 did not flower. One explanation of this would be that they did not receive a sufficient number of naturally inductive days after this date. AccordingIy it appears that plants which received only two or four more inductive nights (moved September g and 11) were able to accumulate 1 enough stimulus to show 75% flowering. The fact that these plants did flower could 1 mean that nights which mav be too short to give full stimulus can contribute a

4 536 CANE BREEDING partial stimulus which is cumulative. This has been shown to be true for other plants?. The frequent occurrence of bunch tops in some plants moved after September 13 is also believed to be an expression of a weak flowering stimulus. All plants which received 4 to 16 fully inductive nights (August 28 to September g) plus whatever nights after September g that were partially inductive showed 100% flowering between October 24 to November 7. The twoplantsmoved to natural day-lengths on September 15 and September 19, and which flowered, could have been in such a position during the night-interruption period that they were shaded and did not receive full inhibition. However their flowering stimulus was weak and associated with this was very late flowering (January 21 and January 27). When the reverse of this experiment was done with H (i.e. plants were moved from natural days to night-interrupted days on alternate days after August 28) There was no flowering but three bunch tops occurred in plants on natural days up to September 17. These results seem to indicate that with plants which receive a sufficient stimulus (such as those moved after September 15) and whickjare then put on inhibitive nights for a long period, there is a dissipation of the stimulus that results in no induction or weak induction. Similar effects have been reported for other plants16. An understanding of these phenomena would be useful in a controlled breeding programme as there are indications that after floral induction has talten place, exposure to long nights will prevent flowering. Further work on partial induction was carried out in the fall of In this test, plants of H in the field were exposed to night-interruption treatments as follows, beginning August I and ending October 15 : (I) interruption every night, (2) interruption on alternate nights, (3) interruption one night followed by two natural nights, and (4) interruption two nights followed by one natural night. Flowering was prevented by all treatments except the third. In this case 20% of the canes initiated flowers and all tassels were very late emerging. While these tests may indicate some accumulation of partial induction in treatment three, the other treatments indicate that if any induction occurred after one natural night, it was dissipated when followed by one or more nights of interruption. Experiments with far-red light were carried out in view of its previously reported effects on sugar cane8 and other plants. An 11.5-hour dark period followed by 4.5 hours of far-red irradiation (735 mp) and then 8 hours of sunlight gave no flower induction. Plants on a 12.5-hour photoperiod were not inhibited by one hour of far-red light (735 mp) during the middle of the dark period, but when a 0.5-hour dark period was interrupted by 0.5 hour of red light (660 mp) followed by 0.5 hour of far-red light (735 mp), no floral initiation took place. However, far-red irradiation of several other short-day plants has reversed red interruptionl1. These experiments would therefore appear to show that far-red light during the dark period does not inhibit floral induction whereas red light does. Also far-red light does not reverse the effect of red light as is reported to occur with several other short-day plantsll. LEAF ACTIVITY AND FLOWERING Experiments have been conducted to evaluate the role of leaves in the biochemical processes involved in the flowering of sugar cane, and also how application of different chemicals to foliage affects this process. Several reports1> 49 6, indicate that such

5 ROBERT E. COLEMAN 537 I compounds as CMU and maleic hydrazide (MH) inhibit or delay flowering. It has also been shown2> 3> that leaf and spindle trimming prevent or delay flowering. To control times of flowering it would help to know exactly the effect of such treatments and when they should be applied for optimum response. Results reported by the Experiment Station of the Hawaiian Sugar Planters' Association2~ 3, indicate that CMU and MH may act differently since MH is more effective earlier than CMU. CMU is more effective after initiation has taken place. Leaf and spindle trimming experiments show that the spindle must be present between August 22 and September 19 for flowering to result and that if the spindle is present for only part of this period flowering is reduced. Apparently some flowering stimulus is still present in the leaves on September 19 and must be translocated from the leaves after that date if flowering taltes place. Other leaf trimming experiments with H confirm that only the upper three leaves and the spindle need be present after September 2 to effect flowering, and the removal of two-thirds of each of these upper leaves during September 2 to 23 completely inhibits flowering. Tests with H and N:Co. 310 show that leaf trimming and spraying $ith MH at the 'flag' stage of flower development completely stop further growth of the tassel. Fourteen-hour day-lengths produced by the extension of natural days with low intensity light had a similar effect on tassel development. ' Leaf and spindle trimming and chemical applications to leaves appear to be means of controlling the time of flowering. There are at present many experiments in progress at this station designed to fix specifically the dates on which these treatments should be applied while at the same time the effects of other newer chemicals are being examined. 1 PRACTICAL ASPECTS OF FLOWER CONTROL I As a result of what has been learned from some of these studies we have been able to recommend and test certain practices which would be useful for controlled breeding. We have repeatedly been able to induce floral initiation in certain varieties by exposing fully matured plants to a fixed 12.5-hour photoperiod in an air-conditioned photoperiod house. If its limitations are talten into consideration, this technique can be of practical value. For instance, night temperatures must be maintained between 70" and 80" F. The plants must be adequately watered, the treatment must be continuous for a period of time and the dark period must be precisely 11.5 hours. With the method we have used (8 hours of sunlight, 11.5 hours of dark, and 4.5 hours of low intensity light) 60 to 90 inductive days are required. This time might be shortened if the plants ~eceived longer periods of full sunlight. Using this method we have induced plants of varieties that naturally flower in November and December during the period April I to July I and used these tassels to malte crosses during the period July 15 to September 19. Similarly photoperiodic induction during the period July I to September 12 has produced tassels for crossing in October. Such crosses as the following have been made by the above technique: H x S.G on July 15, H X Narenga sp. on July 29, H x Glagah Kloet on August g, H x Tainan s9ont. on September 6, N: Co. 31% SES 205A on September 10, N: Co. 310 X E.P.C , H X

6 538 CANE BREI'DINC U.S on September 19, H x E P.C on October 10, N:Co. 310 x P.B on October 18, N:Co. 310 x G oil October 24, and N:Co. 310 x U.S on October 30. Seedlings of these crosses are now in the field awaiting evaluation, but there are indications of a considerable number of true hybrids. As a corrollary to this technique, arrow emergence of carlv flowering varieties might be delayed so that they can be used for crossing wlth othcrs which naturally produce their arrows later in the season. From the experimental work described above we produc 2.1 late tassels of N :Co. 310 by keeping the plants under a treatment of night light init,:luption up to September 15 and September 19. These tassels emerged in late January and were used to make the crosses N :Co. 310 x H on January 21 and N :Co. 310 x H on January 27. In view of these results a large field planting of 25 varieties representing a broad range of flowering characteristics was made. Lights were installed in a field at the Kailua Field Laboratory and the plants exposed to night light interruption,during July I to September 11. As a result of this treatment late flower initiation took place, tassels emerging at a considerably later dat): than those induced under natural conditions. This enabled us to use in crosses tassels of N:Co. 310, Co. 419, Co. 421, H , F , and H in December and January although natural flowering of these varieties occurred in early November. This treatment did not affect the date of tasselling of SES 205A or Erianthus bertgalense and completely inhibited floral initiation in varieties such as B , U.S. 52-9, U.S , H , and some plots of Azul. Two varieties, H and Mol. 6000, initi-.ated flowers after the light treatment, but the tassels did not emerge during the acrossing season. This technique may not be practical for all varieties but there are $enough varieties for which it is effective to make it rather important in controlling certain specific crosses. The use of certain chemicals and leaf trimming may also be effective in controlling flowering, but experimental results thus far at this station have not led to specific recommendations. Compounds suggested by others have been shown definitely to delay flowering, but the periods of delay appear to vary with variety, concentration.of chemical, and the stage of development of the flower initials at time of application. In many cases flower development is delayed to such an extent that the flowers abort and in other cases the tassels which emerge appear abnormal. There is a need for further work in this field. Another technique which may eventually become a tool in a controlled breeding programme is pollen storage. During the past three years experimental work at this station on pollen storage indicated that there are numerous problems associated with such a technique and that practical use of such a method is still in the distant future. Earlier work here in which sugar cane pollen was stored at various temperatures and humidities was not successful as determined by germination tests. Using the vacuum-drying technique described by KING^^ 30 seedlings were produced which we presume to be the products of pollen (H ) stored at room temperature and reapplied to male-sterile tassels of H Four of these seedlings were from pollen stored 7 days. Unsuccessful pollinations were made with pollen stored up to 28 days and kept at 70, 40, and -10" F. Much of our experimental work in developing a technique has been with sorghum

7 ROBERT E. COLEMAN pollen because of its ease of handling. We have found that staining, germination, and the peroxidaze test mentioned by KING^^, and by BUZACOTT and SKINNER= are not reliable. Consequently, we have chosen to measure.the effectiveness of pollen storage by viable seeds produced when the pollen is applied to male-sterile plants, as this is the ultimate criterion of any pollen-storage technique. This method does have some limitations as the male sterility of a 'female' tassel must be assured. Certain problems are associated with the technique of pollen storage. For example, the pollen shed on different days has varying degrees of viability which is either inherent or due to environmental conditions and affects its storage ability. Similarly the conditions of the stigmas when the pollen is reapplied is subject to several sources of variation. While it appears that pollen storage certainly would be an important tool for the plant breeder, further experimentation is needed to develop a practical method. ' SUMMARY Experimental work on both fundamental and practical aspects of a controlled breeding programme is presented. Studies of the photoperiod reactions of sugar cane indicate its extreme sensitivity to exacting requirements for specific periods and sequences of light and darkness. Clonal variations with respect to inductive treatments or senslvity to night light interruption are indicated. Photoperiods which are not optimal may result in a weak flowering stimulus and tend to promote late flowering. Similarly unfavourable lighting regimes following natural inductive photoperiods wlll prevent flowering. While certain chemical and leaf-trimming treatments following floral initiation delay tassel emergence, practical application of such technlques depends upon speclfic knowledge of time of treatment for separate varieties. As a result of increased ltnowledge of some of the factors involved in the flowering of sugar cane, a few techniques can now be used by the plant breeder to make crosses which otherwise might not be possible. Many normally flowering varieties can be brought into tassel at specific times of the year by photoperiodic induction treatments. Naturally late-blooming varieties could therefore be induced for crosses with naturally early-tasselling varieties. The same techniquo could be used to induce naturally early-flowering varieties (which have been inhibited from actually initiating early by night light interruption) to flower late for crossing with regularly late-flowering varieties. Another variation in this technique which has proved to be useful in producing late tassels of a naturally early-flowering variety has been delaying initiation with night light interruption up to a certain date and then allowing the plants to inltiate on the natural days after the light treatments have ceased. Eventually chemical or trimming treatments to the leaves of plants which already contain floral initials may be a method of prolonging or delaying the natural period of tasselling of many varieties. While a reliable and practical method of pollen storage has not as yet been perfected, the potential of such a flexible technique in sugar cane breeding warrants extensive future experimentation. ACKNOWLEDGEMENTS The author gratefully acknowledges the use of the facilities of the Experiment Station of the Hawaiian Sugar Planters' Association in carrying out these studies, and the help of GRAHAM J. DAVIS, formerly plant physiologist with the U.S. Department of Agriculture, who actually conducted some of the experimental work described herein. I REFERENCES 1 Rep. B. W. I. Sug. Cane Breed. Sta., , p Rep. Hawaiian Sug. Ex$. Sta., 1956, p. 50. Rep. Hawaiian Sug. Exp. Sta., I959, p BUZACOTT, J. H., Rep, Bur. Sug. Exp. Stas Qd, 59 : BUZACOTT, J. H., Rep. Bvr. Sug. Ex*. Stas Qd, 60 : 55.

8 54O CANE BREEDING a BUZACOTT, J. H. and SKINNER, J. C., Rep. Bur. Sug. Ex*. Stas Qd, 61 : ' CARR, D. J., On the nature of photoperiodic induction. 111: The summation of effects of inductive photoperiodic cycles. Physiol. Plant., 8 : 512. COLEMAN, R. E., Factors involved in the flowering of sugar cane. Proc. int. Soc. Sug. Cane Tech., 10 : 805. DAVIS, G. J., Artificial induction in sugar cane variety H I.S.S.C.T. Sugar Cane Breeders' Newsletter, 6 : 2. DOORENBOS, J. and WELLENSIEIC, S. J., Photoperiodic control of floral induction. Ann. Rev. PI. Physzol., 10 : 147. l1 DOWNS, R. J., Photoreversibility of flower initiation. Plant Physiol., 31 : 279. la ICING, J. R., The freeze-drying of pollens. Econ. Bot., 15 : 91. la SACHS, R. M., Floral initiation in Cestrunz noctur%um I., a long-short day plant, Plant Physiol., 31 : 185. l4 SALISBBRY, F. B., Photoperiodism and the flowering process. Ann. Rev. PI. Physzol., 12 : 293. l6 SCHWABE, W. W., Evidence for a flowering inhibitor produced in long days in Kalenchoe blossfeldzana. Ann. Bot., Lond., 20 : I. l6 SKINNER, J. C., Delaying arrowing. I.S.S.C.T. Sugar Cane Breeders' Newsletter, 9 : 5. DISCUSSIONS P. C. BRETT (S. Africa) : In one experiment very much more flowering was obtainyd when plants were kept continuously on a IZ+ hour photoperiod than when they were kept first on a 16-hour photoperiod and then subsequently on a 10-hour photoperiod. This does not support the suggestion that sugar cane is a long-short day plant. H. F. CLEMENTS (Hawaii): In my opinion the period before induction had no importance. K. T. GLASZIOU (Australia): I agree with Dr. Clements. Dr. Coleman's experiments do not give sufficient proof to his statement that far-red light does not reverse the effects of red light during the dark period on floral induction. J. T. RAO (India): We have been able to store pollen for 80 days but it was found to lose its fertilizing capacity in 36 hours. EFFECT OF ELEVATION ON ARROWING AND POLLEN FERTILITY IN SUGAR CANE J. F. VAN-BREEMEN, LII- JANG LIU, T. 0. ELLIS and G. ARCENEAUX South Puerto Rico Sugar Company, La Romana, Dominican Republic (Presented by Mr. T.O. Ellis) INTRODUCTION Flowering is a process of vital interest to sugar cane breeders in developing new varieties. The viability of the fuzz produced after fertilization is determined by the same factors that directly or indirectly influence the process of fertilization. COLE MAN^ found that in Hawaii a higher minimum temperature, lesser hours of sunlight, and higher rainfall may have an important effect upon floral initiation. By artificially increasing temperature during tassel development, BRETT~~~ in South Africa was able to obtain a distinctly favourable effect upon subsequent seed setting. This has been confirmed by GRASSL and BELCHER~ at Canal Point, Florida, GARD*+ in Queensland, and PALIATSEAS and CHILTON~ in Louisiana. In 1957 a cane-breeding programme was initiated at Central Romana. A collec-