Shoot Apex Development at Various Stages of Flowering in Sugarcane (Saccharum spp. hybrid)

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1 2008 The Japan Mendel Society Cytologia 73(2): , 2008 Shoot Apex Development at Various Stages of Flowering in Sugarcane (Saccharum spp. hybrid) M. Swapna* and Praveen Kumer Singh Division of Crop Improvement, Indian Institute of Sugarcane Research, Lucknow Received December 26, 2007; accepted June 17, 2008 Summary A study was carried out to understand the flowering behaviour of some commercial sugarcane clones of sub-tropical India and the changes taking place in the target tissues in relation to flowering. The clones studied showed a wide range of flowering behaviour. The shoot apex tissues showed initiation of changes much before the short blade stage. The changes were characterized by an increase in the length and width of the shoot tip tissues and primordial initiation. These changes could be observed 4 5 weeks before the initiation of short blade, which is considered to be an early stage at which flowering could be detected. This gives an idea about specific stages of the crop at which, changes in gene expression patterns during the transition from vegetative to reproductive stage may be observed. Key words Saccharum Sugarcane, Shoot apex tissues, Apical meristem, Vegetative stage, Reproductive stage, Flowering in higher plants comprises of a series of complex developmental processes. The critical point of flowering lies in the changes taking place in the shoot apex tissues during the transition from vegetative to reproductive phase in the plant. Shoot meristem is the source of cells that grow and divide to form all the aerial structures. This also forms the primordia for leaves, inflorescence etc. and is the point of origin of shoot, leaf and floral structures. The conversion of the vegetative meristem to a reproductive one is among the most dramatic examples of a switch in the developmental pathway of higher plants. Flowering in sugarcane is the termination of vegetative growth and has been reported to modify yield in different ways. It might increase or decrease, or have no effect on yield, depending on post-flowering conditions (Hes 1951, Clements 1975, Rao 1977, Gosnell and Julien 1976). But from a breeder s point of view, flowering is essential to recombine alleles and to create variation needed to develop new varieties through conventional breeding. Due to highly heterozygous and complex nature of the crop, as of any other trait, the genetic aspects of flowering have not been studied in detail. Flower induction and the subsequent steps are found to be greatly influenced by environment viz., photoperiod, day and night temperature and by several other factors like maturity of the plant (reviewed in Stevenson 1965, Coleman 1967, Coleman 1969, Moore 1985). Many changes like increase in mitotic activity, increase in total RNA, protein content and ribosomal activity have been reported to be associated with flowering, especially with evocation stage, in various other crops. Temporal and spatial variation in cell number and mitotic activity has been reported in Helianthus (Mare and Palmer 1982). Similar findings have been noted in Lolium (Gonthier and Francis 1989) with a significant increase in mitotic index at particular stages. With the advent of modern molecular biology tools, attempts are being made to identify genes involved in the process of flowering and to understand their role in various stages of reproduction. In sugarcane, James and Miller (James and Miller 1971) studied shoot apex development in early, mid and late sugarcane clones and observed a delay in floral initiation by 1.39 d for a delay of * Corresponding author, sarikkath@yahoo.com

2 174 M. Swapna and P. K. Singh Cytologia 73(2) each day in flowering. They speculated that ratio of width to length of shoot apex may help to detect transition stage up to about one week prior to inflorescence emergence. Julien (1971) observed that for normal flower development in Saccharum clones, a precise sequence of photoperiod is needed. A systematic study regarding very early stages of flowering process like the evocation and other initial processes is still lacking in sugarcane. The present study was aimed at studying the changes taking place in the shoot apex tissues at histological level in relation to the process of flowering, with special reference to very early stages. Materials and methods The plant material consisted of a few commercial clones that had shown a tendency to flower under Lucknow conditions (26 56 N and E at 111 m above sea level) during previous five years (Table 1). No artificial treatments were given to the plants and an attempt was made to study the changes taking place in the shoot apices under natural conditions, at the transition stage and also at subsequent stages. Five shoot tips were collected at regular intervals of one week from each clone and were fixed in Formalin : Acetic acid : Alcohol (FAA-1 : 1 : 18). Tissue fixation in commercial clones was initiated by second week of August and continued up to March. The shoot tips were processed and sectioned using a rotory microtome. These were stained and were observed under light microscope. The median longitudinal section was used for observations. The shoot tip measurements were carried out using ocular micrometer at different stages. Observations were recorded from 20 shoot tips for each stage for each variety during the three years. The sections clearly depicting the changes in shape and structure of tips were photographed. Results In the commercial clones, the flowering pattern was erratic and variable among the clones (Table 1). CoLk 7901 was found to flower regularly during all the years. During and , flowering was observed in CoLk 9618 and CoLk During March 2004, short blade development was observed in a somaclone , Even though tip emergence was observed, there was no complete arrow emergence. The clone CoSe was found to flower profusely during both the years. Thus, wide variation was exhibited among the clones with respect to flowering. Anatomical and histological studies The histological studies were carried out on sections taken using a rotory microtome. The target tissues after fixation in FAA were found to give best results with dehydration using Tertiary Butyl Alcohol (TBA) series, incubation in para-tba at 45 C for 24 h and infiltration in pure paraffin at 60 C for 48 h. Histological studies were carried out in the median longitudinal sections. In all the clones studied it was found that the histological changes are initiated much before the short blade stage, which is considered as an early indication of flowering. The initial changes could be detected by third week of October in a crop planted during first week of March. A typical shoot tip of CoLk 7901 at the vegetative stage is shown in Plate 1a, b. A transverse section at the corresponding stage (Plate 1i) indicates that the classical demarcation of distinct tunica corpus layers is not clear at the initial stages. The apical dome is covered with leaf primordia. The mean shoot tip dimensions are indicated in Table 2. The number of micronuclei was mostly 1 2 at this stage (data not shown). Plate 1c, d depicts a shoot tip of CoLk 7901 that has been fixed at the initial stages of differentiation. This was the earliest stage of evocation that could be identified in this study. The corresponding shoot-tip in the standing crop and a shoot tip separated from the plant for fixation are also

3 2008 Shoot Apex Development In Sugarcane 175 Table 1. Details of flowering in the clones studied Clone Status of flowering Percentage flowering CoLk 7901 Fl Fl Fl CoLk 9618 Fl Fl SB* CoSe NA # Fl Fl NA Nil Nil TE** /9 Nil Nil Nil Fl: Flowered, SB*: Short Blade stage, NA # : Not known, TE**: Tip Emergence. Plate 1 a k. Shoot tip excised from the plant and apical portion after removal of the outer whorls at different stages of plant development. (a, b) Vegetative stage (c, d) Initial stage of transition, showing slight enlargement of the apical portion (e, f) Initial short blade stage (g,h) Advanced short blade stage. i k Longitudinal sections of shoot tip at different stages of development (i) Vegetative stage (j) Initial stage of transition to reproductive stage (k) Advanced stage of differentiation prior to short blade stage, showing initiation of spikelet primordia (sp). Table 2. Mean dimensions of shoot tip (length and width) at different stages Clone * Stage I ** Stage II *** Stage III L (m) W (m) L (m) W (m) L (m) W (m) CoLk CoLk CoSe / Mean CV% * Stage I: Vegetative Stage, ** Stage II: Transition stage, *** Stage III: Differentiation stage preceding short blade stage.

4 176 M. Swapna and P. K. Singh Cytologia 73(2) shown. Internodal elongation is very prominent at this stage. Even though external symptoms of flowering are not visible at this stage, the shoot tip shows a slight bulging at the base, once the layers surrounding the apical dome are removed. The apex tissues at this stage show an increase in dimensions with a mean height of m and width of m. The apical dome shows signs of enlargement with primordial initiation being prominent in the sections. (Plate 1j). The intensity of staining is more in the periphery, indicating active division in the peripheral cells. The shoot apex continues its growth. This is the stage just before the short blade emergence (Plate 1e, f). The initiation of inflorescence branch primordia is clearly visible at this stage (Plate 1k). By the time short blade emergence is observed, the internal modifications have advanced further. The spikelet formation becomes visible. The prime target tissues, where the changes are concentrated prior to flowering, lie towards the axillary bud sites in the spikelet primordia. The target tissues can be considered as those meristematic tissues, which in response to the floral stimulus specifically initiate floral morphogenesis. The mean length of the apical portions increases, with a decrease in the width (Table 2). Thus, during the early stage of transition, the length and width of shoot apex increases (Table 2, Stage I to Stage II), with the width showing a reduction as the plant approaches the short blade stage (Stage III). Discussion The success of any crop improvement programme depends on the regular flowering of parental lines involved. Wide variation is observed in the intensity/extent of flowering in sugarcane, even in tropical conditions, where it flowers naturally. The same applies to sub-tropical situations also and in general, flowering is almost completely absent. A systematic study of the flowering pattern in this crop and the changes associated in the target tissues may help in the long run, in further basic studies related to this phenomenon. From the sections taken at these stages it can be noted that the internal changes leading to flowering are initiated much before the short blade stage. 4 5 weeks before the short blade is visible, the shoot tip sections show a transition from the leaf apex to an inflorescence apex (Plate 1j). Probably, this may be one among the initial stages at which, one can look for changes in gene expression patterns that governs vegetative-to-reproductive phase transition. This information assumes significance, even in the present era of advancements in the field of molecular and cellular biology (Ming et al. 2002). Also an idea about the earliest stages of reproductive primordia induction/initiation aids in pin-pointing the exact stages at which artificial treatments for manipulation of flowering process can be attempted. Even though such procedures are already being put into practice by sugarcane workers abroad, in India, and especially in the sub-tropics, such studies are yet to be initiated seriously. Especially in the context of environmental changes taking place globally, situations may arise where novel possibilities and target areas have to be explored for induction/utilization for sugarcane flowering and hybridization. Such basic information can be a stepping stone in a broader perspective of reproductive manipulation. Even though there is variation among the clones with respect to the dimensions of shoot apex at histological level at different stages, the extent of variation appears to be less in the case of width than in the case of shoot apical length. This points towards the possibility of width being a better indicator of transition from vegetative to reproductive stage than length. The ratio of length to width of shoot tip did not exhibit a significant relationship with the transition stage, at this early stage of differentiation. Such a relationship was observed by earlier workers (James and Miller 1971) at later stages of flowering. Also, in some of the sections in CoLk 9618 there was an increase in the number of micronuclei as the shoot tip advances form vegetative (average of 2 micronuclei) to reproductive stage (average of 4 micronuclei). But this could not be confirmed in other genotypes.

5 2008 Shoot Apex Development In Sugarcane 177 One of the early events reported in many crops, following shortly after evocation is an increase in mitotic index in the apical cells (Bremer 1970). But a conclusive evidence for such an increased mitotic index at the apex tissues could not be observed in the present study. There was clustering of cells at the periphery with a large number of dividing ones, but the observation was not uniform in all the sections studied. In Lolium an absence of an early mitotic rise has been reported. The absence of cells in the appropriate mitotic stage had been cited as a probable reason in this species (Bremer 1970). The same argument may hold good in the case of sugarcane also, which also belongs to the grass family. The investigation gives a basic idea about the different stages in the process of vegetative-toreproductive transition and subsequent stages of flowering in sugarcane. The extent of variation in the different genotypes under the same environment with respect to flower initiation and subsequent advancements, and the variation in flowering in the same genotype during subsequent years, compels one to be cautious in one s approach while studying the functional and regulatory mechanisms of flowering in this crop. This study also gives an idea about specific stages of the crop, at which changes in gene expression patterns during the transition from vegetative to reproductive stage may be observed. Acknowledgements The authors acknowledge the help rendered by scientists in different divisions of the institute, during various stages of the study and also in providing facilities for photomicrography. References Bremer, G Structural and metabolic changes in the shoot apex in transition to flowering. Can. J. Bot. 49: Clements, H. F Flowering in sugarcane: mechanisms and control. Hawaii Agric. Exp. Stn. Tech Bull. 92: pp 56. Coleman, R. E Some aspects of flowering stimulus production in sugarcane. Proc. Int. Soc. Sugar Cane Technol. 12: Physiology of flowering in sugarcane. Proc. Int. Soc. Sugar Cane Technol. 13: Gonthier, R. and Francis, D Changes in pattern of cell division in the shoot and root meristem of Lolium perenne during transition from vegetative to floral growth. J. Exp. Bot. 40: Gosnell, J. M. and Julien, M. H. R Variations in the effects of flowering on cane yield and quality. Proc. Sugarcane Ripeners Semin., Monsanto 1: Hes, J. W The effect of arrowing on yield and quality of sugarcane. Sugar J. 14: James, N. I. and Miller, J. D Shoot apex development in early-, mid- and late season flowering sugarcane clones. Proc. Int. Soc. Sugar Cane Technol. 14: Julien, M. H. R The photoperiod control of flowering in Saccharum Proc. Int. Soc. Sugar Cane Technol. 14: Mare, J. and Palmer, J. H Changes in mitotic activity and cell size in the apical meristems of Helianthus annus L. during transition to flowering. Am. J. Bot. 69: Ming, R., Del Monte, T. A, Hernandez, E, Moore, P. H., Irvine, J. E. and Paterson, A. H Comparative analysis of QTLs affecting plant height and flowering among closely related diploid and polyploidy genomes. Genome. 45: Moore, P. H Saccharum. In: A. H. Halevey (ed.) CRC Handbook of Flowering. Vol. 4. CRC Press Inc., Boca Roton, Florida. pp Rao, P. S Effects of flowering on yield and quality of sugarcane. Exp. Agric. 13: Stevenson, G. C Flowering in sugarcane. In: Genetics and Breeding in Sugarcane. Longmans, London. pp