Cytogenetic Analysis of Thermosensitive Genic Male Sterility (TGMS) Recovered from a Pennisetum glaucum (L.) R. Br. P. violaceum (Lam.) L.

2004 The Japan Mendel Society Cytologia 69(4): 409 418, 2004 Cytogenetic Analysis of Thermosensitive Genic Male Sterility (TGMS) Recovered from a Pennisetum glaucum (L.) R. Br. P. violaceum (Lam.) L. Rich Cross Pankaj Kaushal*, Ajoy K. Roy, Surinder N. Zadoo and Rang. N. Choubey Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi 284 003, India Received January 30, 2004; accepted August 14, 2004 Summary One male-sterile plant was recovered from a cross between pearl millet (Pennisetum glaucum) and its wild progenitor P. violaceum. The behavior of male sterility was temperature dependent, as the development of pollen mother cells to pollen grains was found to be dependent on temperature. The plant showed sterility at lower temperatures while it was fertile at higher temperatures during seasonal variations. This thermosensitive male sterility trait was found to be genetically controlled based on the segregation data of backcross generation. Segregation was observed for ca. 50% of the population expressing this trait. These TGMS plants exhibited transition from fertility to sterility when mean daily temperature reduced to ca. 20 C or below, and fertility was restored above this temperature. The period of 4 to 5 d prior to anthesis was found to be most sensitive to temperature variations. Chromosomal studies on the parent plant as well as segregants showed 2n 14 with regular meiosis during non-sensitive phase (period exhibiting male-fertility) ruling out aneuploidy and/or gross chromosomal structural aberrations as a cause of male sterility. Fertility characters were also studied for TGMS and non-tgms plants during sensitive and non-sensitive phases. Pollen grains shed from the TGMS plants were much more vulnerable than from normal plants during sensitive phase indicating poor development of the membrane and walls of the pollen grains during sterility inducing conditions. Transition characteristics from male sterility to male fertility during sensitive and non-sensitive phases for TGMS and non-tgms plants are discussed. Key words Environmental Sensitivity, Mutations, Male Sterility, Pearl Millet, Wide Hybridization. Environment sensitive male sterility is a system where the sterility reaction is controlled by extreme environmental conditions. It has many advantages over other male-sterility systems as no restorer-breeding programme is needed and the male sterility can be utilized for hybrid seed multiplication in a specific environment. Two major classes of such sterility, viz., photoperiod sensitive male sterility (PSMS) and thermosensitive male sterility (TMS) have been reported in crops like rice (Mei et al. 1999), wheat (Murai and Tsunewaki 1993), Petunia (Van Marrewijk 1969), sorghum (Brooking 1976) and barley (Gupta and Singh 2000). TGMS systems have been extensively studied in rice and 6 TGMS genes (tms1, tms2, tms3, tms4, tms5, ms-h) have been reported and molecularly mapped so far (reviewed in Wang et al. 2003, Lopez et al. 2003). Pearl millet (Pennisetum glaucum) is an important crop utilized world wide as a food and fodder crop. Five cytoplasmic male sterility (CMS) systems are mainly used in breeding programmes. In addition to A 1, A 2 and A 3 CMS sources (Burton and Athwal 1967) used in hybrid breeding, 2 other sources (A 4, A v ) derived from different accessions of P. violaceum (Marchais and Pernes 1985, Hanna 1989), have been reported. P. violaceum being sexually compatible with pearl millet is an important source of stable male sterility-inducing cytoplasm (Rai et al. 1996). In an attempt to study responses of embryos of interspecific cross (P. glaucum P. violaceum) to the process of embryo cloning, we recovered a thermosensitive male-sterile plant from cultured * Corresponding author, e-mail: pkaushal@rediffmail.com or pankaushal@igfri.up.nic.in

410 Pankaj Kaushal et al. Cytologia 69(4) zygotic embryos. The plant showed considerable variation from male-sterility to male-fertility that could be correlated to temperature variations during seasonal changes. In the present communication cytological and genetical characteristics of this thermosensitive male sterile plant and segregation of the trait in subsequent generations and its genetic nature is discussed. Material and methods The experiment was carried out at the Indian Grassland and Fodder Research Institute, Jhansi, India (25 27 latitude, 78 35 longitude), during 1998 to 2000. Recovery of a thermosensitive male-sterile plant Artificial pollination was attempted between pearl millet male sterile line IP 17963 (with A 1 male sterile cytoplasm) and P. violaceum (IP 21737), procured from ICRISAT, Hyderabad, India. Developing embryos were cultured 13 14 d after pollination following Mohindra (1989). Twenty plants could be successfuly transferred to field 15 d after germination. All the embryo-cultured plants except 1 were normal in their characteristics and seed set. However, one plant (F1PV-1) showed thermosensitive male sterility. All plants were studied for their morphological, reproductive and cytological characteristics and a comparison was made between F1PV and its siblings. Selfed and crossed seeds were produced on this plant. Characterization of male sterility This thermosensitive male sterile plant was backcrossed as maternal parent with line IP 17964, the maintainer of the male-sterile line IP 17963, to obtain back-cross generation (BC1) during winter season when the plant F1PV-1 showed male sterility. These seeds were sown in normal season in July 1999. Standard agronomical practices were followed for irrigation and fertilizer application. Parental lines IP 17963, IP 17964 and P. violaceum were also grown as controls. Plants belonging to BC1 generation initiated flowering on Oct. 20, 1999. Emerging spikes were bagged with glycine bags to prevent outcrossing and to observe selfed seed setting. Data on pollen fertility and other aspects were recorded on 210 BC1 plants till April 2000. Observation on fertility/sterility was recorded at 2 weeks interval (hereafter called week-interval) and 3 4 spikes were tested per plant and averaged. Plants were classified as sterile or fertile by the absence/presence of pollen dust in selfing bags, presence of pollen clouds during anthesis time (900 1100 h) as well as formation of seeds by self-pollination and then compared with the seasonal variation. Cytological characterization of the requisite plants was made on pollen mother cells (PMCs). Young panicles were fixed in aceto-alcohol (1 : 3) at appropriate stage and anther squash preparations were made in aceto-carmine stain (1% or 2%). Data was recorded for chromosome number and pollen fertility. Well-filled and stained pollen were treated as fertile, whereas, empty, shrivelled and partially stained pollen were treated as sterile. Cytological studies were conducted on material fixed at critical intervals to observe temperature effects on developmental stages of pollen mother cells as well as its relation in governing male-sterility or its restoration. Data on daily temperature was obtained from the Meteorological Observatory of the Institute. Results and discussion F1PV-1 plant All the embryo-culture derived plants from pearl millet P. violaceum cross were normal in their characteristics and set seed, except F1PV-1 which behaved as thermosensitive male sterile. This plant exhibited male sterility at lower temperatures during November to February (1998 1999) (average daily temperature 13.5 20.2 C) whereas at higher temperatures during March April

2004 TGMS in pearl millet 411 Table 1. Fertility characteristics of thermosensitive male-sterile plant F1PV-1 Year Time period (date/month) Temperature ( C) Range Average (min. max.) Spike emergence (no.) % stainable pollen Cross Seed set Self 1998 4/11 17/11 19.55 13.5 30.5 2 3 23 0 18/11 1/12 19.1 8.0 29.0 5 5 46 0 2/12 15/12 17.3 6.6 28.9 6 0 38 0 16/12 31/12 15.17 5.6 24.3 3 0 44 0 1999 1/1 14/1 13.72 5.4 24.5 4 2 27 0 15/1 28/1 13.5 3.9 24.9 4 1 23 0 29/1 11/2 14.42 5.7 22.3 5 16 41 0 12/2 25/2 20.02 10.0 30.5 7 25 37 7 26/2 10/3 22.42 10.7 35.2 9 53 40 15 11/3 24/3 22.32 10.5 34.5 8 62 31 28 25/3 7/4 27.35 13.5 40.0 9 76 33 25 8/4 21/4 29.3 17.4 41.2 6 63 32 26 22/4 5/5 33.65 20.8 44.9 7 65 29 16 6/5 19/5 33.52 24.0 42.2 6 60 22 14 20/5 2/6 33.22 25.5 41.3 5 67 12 9 3/6 16/6 32.79 24.5 40.0 2 68 7 6 17/6 30/6 33.65 27.8 39.6 2 62 3 2 Table 2. Morphological characteristics of thermosensitive male-sterile plant F1PV1 Character Male sterile plant (F1PV-1) Normal plants (siblings) Plant height 108 cm 104 10.2 Leaf length 33 cm 35.2 4.3 Leaf width 1.3 cm 1.2 0.3 No. of nodes 6 6.7 1.0 Internodal length (2nd and 3rd node) 30 cm 29.6 3.1 Tiller number 15 8.3 3.6 Stem thickness 0.9 cm 0.7 0.1 Spike length 10 cm 8.8 1.5 Spike width 1.2 cm 1.1 0.3 Days to flowering initiation 63 d 60.9 4.5 Anther colour- Sterile-purple Fertile-Yellow Fertile-yellow Spike colour Purple Purple Stigma colour White White (1999) and onwards it behaved as male fertile. It was observed that the male fertility was restored between 20 and 25 C. Their fertility behaviour and morphological characteristics of the plant are presented in Tables 1 and 2. This plant showed better primary and secondary tillering, winter hardiness, and greater longevity than other siblings. It completed duration of 275 d of life cycle from September 1998 to June 1999. The plant showed male sterility until the second week of February (average temperature 20 C). Later it recovered fertility with the rise in temperature. A few stainable pollen grains observed in January 1999 were highly sticky and fragile and were believed to be operationally nonfunctional. It was interesting to note that during sudden rise in average temperature for 4 d during the third week of February, spikes showed transition from sterility to fertility for a short period and few spikes were seen having sectors of both sterile and fertile anthers. Female fertility remained

412 Pankaj Kaushal et al. Cytologia 69(4) unaffected as seeds from cross-pollination could be obtained throughout the flowering period. Reduced seed set in June in spite of higher pollen stainability could be attributed to poor receptivity of stigma due to higher temperatures. The crossed seeds obtained from this plant were healthy and well filled, whereas viable selfed seeds could not be obtained. Cytologically, the plant exhibited 2n 14 chromosomes as identified through meiotic preparations. Regular meiosis was observed in the pollen mother cells (PMCs) in warmer months. This ruled out the probability of chromosomal interchanges or other chromosomal aberrations including aneuploidy as the cause for male sterility. Regular metaphase and equal chromosome segregation at anaphase was observed in these cells. In colder months anthers were characterized by hollow sacs with no PMCs. With the increase in seasonal temperature, formation of PMCs was induced but Fig. 1. Cytological observations in thermosensitive male-sterile plant. a, hollow anther/pollen sac during colder months; b, pollen grain unable to withstand 1% acetocarmine osmoticum; c d, sticky and fragile pollen grain, produced during intermediate months; e f, variable size viable pollen grains produced during warmer months; g h, regular meiosis with 2n 14 chromosomes exhibited by PMCs during non-sensitive phase; g, diakinesis with 7II, h, anaphase with regular 7 : 7 chromosome distribution.

2004 TGMS in pearl millet 413 their development was arrested at earlier meiotic stages (Fig. 1). A synchrony was observed between rise in seasonal temperature and increasing regularity in meiotic divisions. It appeared that male fertility change was controlled by temperature during the meiotic division of pollen mother cells, about 4 5 d (Fig. 2) before heading and that the temperature inducing the male fertility change was somewhere between 19 21 C. Fig. 2 also shows the effect of a sudden increase in temperature during late February reflected 4 5 d later in spikelet fertility. Rise in temperature during February March resulted in highly Fig. 2. Spikelet fertility in response to fluctuating daily temperature in the plant F1PV1. fragile and sticky pollen which were seen in lumps of 30 35 and usually burst even in 1% acetocarmine staining solution (Fig. 1). Average pollen stainability in colder months was 1.8% as compared to 65.4% in warmer months. Self-pollination did not yield seed set until late February. During March, and onwards, the meiosis was regular and well stainable but variable sized pollen were produced. Remaining plants were normal in their cytological behaviour and exhibited regular meiotic stages and formation of fertile pollen grains at all seasonal temperatures. The phenomenon of environmentally influenced male sterility has been previously reported in pearl millet (Mashingaidze and Muchena 1982, Rai and Hash 1990, Rai et al. 1996) from various combinations of male sterile and maintainer lines. Some of these investigations used P. violaceum cytoplasm (i.e. maternal parent) as a source of new cytoplasmic male sterility (Marchais and Pernes 1985, Hanna 1989). The present report is perhaps the first report of recovery of environmentally sensitive male sterility in pearl millet obtained using P. violaceum as male parent. We assume that this phenomenon might have occurred due to some spontaneous or in-vitro mutation, as the immature zygotic embryos from pearl millet and P. violaceum cross were passed through a tissue-culture phase during the development (Shi 1985, Jain 2001). To test the genetic control of sterility of this plant, BC1 generation was raised and studied. Back-cross generation (BC1) Segregation for thermosensitive male-sterility The genetic nature of the thermosensitive male sterility was studied using 210 BC1 plants in 1999. The plants were found to segregate for this trait (Table 3), hence, a genetic origin (nuclear) for the trait is suggested. The fertility expression in relation to the seasonal temperature was the main criteria for classification of plants as possessing thermosensitive male-sterility trait (hereafter called as thermosensitive genic male sterility TGMS plants) or not possessing it (non-tgms plants). Flowering initiation was observed on Oct. 20, 1999 ca. 10 weeks after sowing. All plants, except two, were initially fertile as evident from the data on selfed seed setting and observations on pollen clouds/dust during anthesis. During the week-interval 2 (Nov. 5 18, 1999) 15.7% sterile plants were observed which further increased to about 50%. This proportion of sterile plants remained constant till further rise in temperature during March 12 25, 2000 (week-interval 11), where we again observed decrease in ratio of sterile plants to 19.5%, and in the following week only 2 sterile plants were observed. Chi-square analysis of sterile and fertile plants during sensitive phase fits highly significantly to 1 : 1. The period could be divided into non-sensitive, sensitive and transition phases depending upon the male sterility reaction (Table 3).

414 Pankaj Kaushal et al. Cytologia 69(4) Table 3. Proportion of fertile and sterile BC1 plants responding to seasonal temperature fluctuations Time period Week interval Range Average daily Fertile plants Sterile plants Phase (1999 2000) (WI) (min max) temp. ( C) (F) (S) designated 22/10 4/11 1 13.9 32.3 23.47 208 2 Non-sensitive 5/11 18/11 2 10.5 31.8 21.6 208 33 Transition 1 19/11 2/12 3 8.6 28.1 18.4 108 102 Sensitive 3/12 16/12 4 6.9 26.0 16.35 109 101 Sensitive 17/12 31/12 5 5.1 24.6 14.42 103 107 Sensitive 1/1 14/1 6 5.0 26.5 15.32 100 110 Sensitive 15/1 28/1 7 4.6 25.1 15.1 101 109 Sensitive 29/1 11/2 8 7.5 25.6 16.2 100 110 Sensitive 12/2 25/2 9 4.8 25.3 15.02 102 108 Sensitive 26/2 11/3 10 9.9 30.4 20.32 103 107 Sensitive 12/3 25/3 11 10.1 31.8 21.35 207 41 Transition 2 26/3 8/4 12 13.1 38.4 25.3 208 2 Non-sensitive Transition phases 1: male fertility to male sterility, 2: male sterility to fertility. Certain plants were observed to express TGMS trait approximately 4 weeks after flowering initiation in week-interval 2 which continued for an interval of ca. 16 weeks (week interval 10). From week-interval 2, with the reduction of daily temperature TGMS plants showed transition towards male sterility and eventually the selfed seed setting reduced to 3.2% in following week. The average selfed seed setting during the sensitive phase was 2.3% with a lowest value of 0.3% in week interval 7 when the average temperature dropped to 15.1 C. Mean temperature has been used to denote the critical sterility/fertility points in several studies (Zhang et al. 1991, Lu et al. 1994, Shen et al. 1994, Tong-Min et al. 2001) whereas in many reports major role of maximum (Rai and Hash 1990, Viraktamath and Virmani 2001) or minimum temperature (Brooking 1976) has also been advocated. In present study the transition of plants between fertility and sterility revealed that temperature below 21 C was inducing the male sterility (in TGMS plants) whereas temperature above 21 C reverted to male fertility. However more experiments are required to define the contributing components for the critical average temperature. Two BC1 plants were found to remain sterile throughout the period of study. Cytology, pollen fertility and seed setting Randomly selected 26 BC 1 plants showed 2n 14 chromosomes with regular meiotic divisions during non-sensitive phase, thereby confirming the absence of chromosomal aberrations as cause of male sterility. The anther plumpness and pollen shedding in this study was taken as a measure of pollen fertility and shrunken anthers with lacking pollen shedding as measure of sterility (following Rai and Hash 1990). All the plants identified as TGMS showed highly reduced fertility as expressed by selfed seed set (2.3 1.4) and pollen fertility (4.5 1.2) during the sensitive period, as compared to selfed seed-setting (54.1 3.8) and pollen fertility (75 2.8) of non-tgms plants. For non- TGMS plants, no difference was observed for selfed seed set during sensitive and non-sensitive phases (Fig. 3). Fig. 4 represents a comparison of fertility characteristics of TGMS and non-tgms BC 1 plants during sensitive and non-sensitive phases. Control plants were also unaffected in seed setting and pollen fertility by the seasonal fluctuations. Differential staining showed that the sterile pollen grains were shrivelled, degenerated, vacuolated and devoid of starch and cytoplasm. In contrast, viable pollen grains had dense cytoplasm and were packed with starch grains. TGMS plants (F1PV-1 as well as BC1 segregants) shed some pollen near to the critical temperature which under microscopic examination ruptured in even 1% acetocarmine, while the pollen from non-tgms plants during the same period as well as pollen from TGMS plants in non-sensi-

2004 TGMS in pearl millet 415 Fig. 3. Seed setting (%) in TGMS and non-tgms plants in relation to seasonal temperatures. Fig. 4. Comparison of fertility characteristics of TGMS and non-tgms BC1 plants during sensitive and non-sensitive phases. tive phase could withstand at least 2% acetocarmine indicating vulnerability of pollen to the osmoticum. This might be due to poorly developed plasma membrane. Shen et al. (1994) observed similar deformities in environmentally sensitive male sterile mutant of rice. Tong-Min et al. (2001) considered that among environmental-sensitive genic male sterile lines in rice, the sensitive stage was pre-meiotic division stage or filling of pollen grains. Results of the present investigations on synchronous meiotic development with increasing temperature could be explained likewise. Furthermore, under male-sterility inducing conditions, the development of the plasma membrane and the walls of the pollen Fig. 5. Pollen-fertility of TGMS and non-tgms plants in response to fluctuating daily temperature during transition phase. grains from the mutant were hampered in addition to the inhibition of starch filling, thus resulting in non-functional and abortive microspores. The stages from formation of pollen mother cell to late uni-nucleate of pollen grains were the most sensitive to temperature. In general, all the plants produced variable sized pollen when at maturity stage. Though interspecific incompatibility does not exist (Kaushal and Sidhu 2000) and the 2 species are sexually compatible, we experienced different genotypic combinations of these species producing variable size pollen grains explainable due to differences in starch accumulation. Partial reproductive barrier between some combinations of these species have also been reported (Amoukou and Marchais 1993). To observe the effect of daily temperature and to find the developmental stage affected by temperature, we studied the pollen fertility of randomly chosen TGMS and non-tgms plants (5 plants from each category) during the transition phase (Fig. 5). Results indicated that the plant response to low temperature was dependent on the developmental stage of the young panicle. The observations were similar to the pollen development in plant F1PV1, where the pollen fertility was affected by the temperature at the time of heading, i.e. 4 5 d prior to anthesis (Fig. 2). This was, however, interesting to note that temperature higher than presumed critical temperature during Nov. 15 16, 1999

416 Pankaj Kaushal et al. Cytologia 69(4) ( 22 C) has restored partial fertility approximately 4 5 d after spike emergence, and again reduction in temperature yielded reduction in pollen fertility (Fig. 5). Thus, in the present study, the most sensitive stage appeared to be 4 5 d before anthesis. Earlier similar studies reported period of 4 to 8d after panicle initiation as stage most sensitive to temperature variations (Maruyama et al. 1991, Borkakati 1994, Viraktamath and Virmani 2001), though it has been found to be influenced by the genetic background (Tong-Min et al. 2001) as well as modifier genes (Rongbai and Pandey 1999). Several TGMS plants were observed with fertile and sterile sectors specially during week interval 3 in BC1 population similar to our observation made in F1PV-1. It was found, for example, on the first day of anther emergence on a panicle, shriveled indehiscent anthers emerged and there was no pollen shedding, but on the 4 or 5 d anthers shedding an abundance of pollen emerged on other sections of the panicle. The main reason for this could be that field temperatures were fluctuating and spikelets at different positions on the same panicle were at different developmental stages at any one time. As the critical stage was very short and any shorter period of exposure to low temperature would induce sterility in only part of the inflorescence. Thus because of temperature fluctuations some spikelets that experienced temperatures at or below critical temperature during the short critical period were completely male sterile. At the same time, other spikelets on the same panicle that were formed before or after the critical stage were not affected and they produced anthers that shed some pollen if they experienced temperatures above the critical temperature. Similar observations were reported by Mashingaidze and Muchena (1982) in pearl millet and by Downes and Marshall (1971) and Brooking (1976) in sorghum. TGMS is nuclear genetic The population study of TGMS trait in BC1 generation yielded 1 : 1 segregation for plants with and without TGMS trait suggesting its nuclear genic control. This segregation ruled out the possibility for this trait being cytoplasmic or maternally inherited as well as under complete control of environment or as an adaptive response to extreme environmental condition. Association of wild genome (P. violaceum) within cultivated cytoplasm has been reported to maintain male fertility (Marchais and Pernes 1985) and transfer of wild cytoplasm to cultivated genetic background has produced male sterile lines (Hanna 1989). Marchais and Pernes (1985) also obtained sterile plants wherein P. violaceum was used as female in cross with P. glaucum whereas P. violaceum as male parent resulted in fertile plants. Except F1PV-1 all the F1s in present study were fertile showing P. violaceum could restore fertility in such crosses. Fertility restoration genes were already reported to be present in P. violaceum (Marchais and Pernes 1985). The fertility expression of plants with restorer genes in sterilizing cytoplasm is also reported to be influenced by environment, especially temperature (Van Marrewijk 1969). Thus, one of the possibilities for segregation of TGMS trait could be the differential expression of restorer genes from P. violaceum in P. glaucum background in BC1 generation. Cytoplasmic male sterility is by far the most widely used system for developing pearl millet hybrids. CMS system, although stable, is cumbersome to use for developing hybrids. It restricts the use of varieties/elite lines as parental lines for requirement of specific maintainer and restorer genes, and there is potential risk of genetic vulnerability caused due to possible association of a CMS system with susceptibility to a biological stress. TGMS system can be useful in the tropical countries where temperature variations between the altitudes/seasons are available. Vikartamath and Virmani (2001) have already discussed many benefits of deploying TGMS system for developing hybrids. Stabilization of such TGMS lines in pearl millet might be helpful in breeding hybrids as well as for study of genes responsible for induction of male sterility under extreme environmental situations.

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