Abstract: An experiment comprised of 5 male sterile lines and 30 inbred testers of pearl millet and 150 hybrids was conducted at Millet Breeding Station, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University, Coimbatore during 2005-2007. The fertility restoration ability of thirty diverse inbred pollinators on different cytoplasmic sources were studied. Among the one hundred and fifty hybrids studied, forty one hybrids were complete sterile and twenty four hybrids were recorded fertile. The two inbreds namely PT 5259 and PT 5773 contributed sterility with four male sterile lines out of five male sterile lines used. The former expressed sterility with ICMA 88994A, 81A, ICMA 94111A and PT 5054A, the later expressed sterility with the male sterile lines 81A, 732A, ICMA 94111A and PT 5054A. The A line PT 5054A expressed sterility in fifteen hybrids out of inbreds studied and it was followed by 81A with in fourteen inbreds. The 732A cytoplasm expressed sterility with only one inbred (PT5773) and also it was registered complete fertility with most of the inbreds than other male sterile lines. Developmental variation in the male sterile and male fertile lines of pearl millet was studied in five developmental stages viz., PMC, dyad, tetrad, soon after microspore release and before dehiscence of anther and it was found that, break down of fertility in the sterile anthers of pearl millet was post meiotic, in specific soon after the release of tetrad in the developmental stage.
INTRODUCTION
Taxonomically pearl millet (Pennisetum glaucum (L.) R.Br.) belongs to the family Poaceae, sub-family Panicoideae, section Penicillarium and possesses 2n = 2x = 14 chromosomes. It is a cross-pollinated annual C4 crop species that originated in western Africa and was introduced to eastern Africa and the Indian sub continent some 2000 years ago. India is the largest producer of this crop, both in terms of area (9.1 m ha) and production (7.3 m t), with an average productivity of 780 kg ha-1 during the last five years. Nearly 50% of the millet area is under hybrid cultivars. Earlier reports of occurrence of male sterility in pearl millet were by Kajjari and Patil (1956) and Burton (1958). The discovery of cytoplasmic male sterility in pearl millet (Burton, 1958), led to the production of high-yielding commercial hybrids using the male sterile line, Tift 23A1. Subsequently, two additional sources of cytoplasmic male-sterility designated L66A2 and L67A3 were identified (Burton and Athwal, 1967). In fact, a number of male-sterile lines were developed by genome substitution of Tift 23A1 by repeated back-crossing to several maintainers (Burton, 1965; Pokhriyal et al., 1974; Athwal et al., 1976). The use of male sterile line Tift 23A, in producing high yielding hybrids failed to last long due to downy mildew epidemics. Hence, to obviate the dangers of genetic uniformity and disease susceptibility, attempts have been made in pearl millet to intensity the research on cytoplasmic and genetic diversification of male sterile lines (Kumar and Andrews, 1984; Rai and Singh, 1987). Both nuclear and cytoplasmic diversity will have practical utility in exploitation of heterosis. Altering the male sterile lines by incorporating the nuclear genome of alternate maintainer lines may lead to genetic and cytoplasmic diversity, thereby providing a wider base for further exploitation. With the availability of number of male sterile lines in diverse cytoplasmic background, the scope for developing superior hybrids has enlarged. However, information on the performance of these male sterile lines in combination with specific restorer is a pre-requisite in selecting suitable parents for the yield and its contributing characters to develop a high yielding commercial hybrid.
Comparative studies of pollen sterility/fertility and meiotic studies in four temperature-sensitive genic male sterile (TGMS) lines, along with restorer line RHRBI-458 of pearl miller, were conducted studied by Shinde and Mehetra (2003). The TGMS lines (RHRBTS-9862, RHRBT-9865, RHRBT-9869 and RHRBTS-9872) had major portion of their anthers is completely sterile class (97.8 to 99.7% under high temperature conditions of >38.7° PMC of earlier tillers), while the major portion of their anthers was classified into completely fertile class (from 97.7% in low temperature of <34.2°). The fertile line RHRBI-458 had 99.1 to 100% completely fertile anthers under both environments.
Microsporogenesis of cytoplasmic male sterile lines and male fertile lines was studied in order to identify the stage of breakdown of fertility during microsporogenesis which leads to male sterility in pearl millet. Fertility/sterility pattern of hybrids (Rai and Hash, 1990) and mitochondridal DNA-RFLP pattern (Chhabra, 1995) have been used to characterize these CMS sources. Microsporogenesis pattern has been used to characterize CMS differences in maize (Lee et al., 1979). Pearl millet studies of A1 system CMS lines show strong effect of the genetic background on microsporogenesis. For example, Burton (1958) indicated that microsporogenesis breakdown occurs mainly during tetrad formation. Influence of a range of cytoplasm on microsporogenesis and anther development in pearl millet using six isonuclear A-lines having five cytoplasm (A1,A2,A3,A4 and A5) and the nuclear genome of 81 B were studied by Chhabra et al. (1997).
MATERIALS AND METHODS
Five male sterile lines and thirty inbreds of pearl millet were chosen for
the study. The materials were obtained from pearl millet unit, Department of
Millets, Centre for Plant Breeding and Genetics, Tamil Nadu Agricultural University,
Coimbatore. Among the five male sterile lines used as female parents, ICMA 94111A
and 81A are having A1 cytoplasm, ICMA 88004A and PT 5054A have A4
cytoplasm and 732A belongs to Bellary cytoplasm, a different source which
had been derived from PT 819, an inbred line originated from Andhra Pradesh,
India. The thirty inbreds were used as testers. Artificial selfing and crossing
operations were carried out. To ensure selfing, the panicles of the parents,
just before the emergence of stigma were covered with butter paper bags and
fastened well with a piece of twine at the base to prevent contamination. After
complete protrusion of stigma, the panicle of the seed parent was pollinated
with pollen collected from the desired male parent and again covered. Pollination
was effective between 8.00 to 11.00 am. The resultant hybrids along, their parents
were raised in Randomized Block Design with three replications during Kharif
2006. Each hybrid was accommodated in one row of 45x15 cm. Uniform and recommended
cultural practices were followed. At 75% anthesis, plants were scored for pollen
shedding in early hours between 7.00 to 9.00 a.m. by tapping the unbagged ears
and observed for pollen cloud. In all fertile/sterile plants anthers from florets
that will open on the following day were collected and squeezed with a needle
in a drop of acetocarmine 2% stain and studied under microscope. Completely
round and well stained pollen grains were counted as fertile while, the shriveled,
unstained or partially stained ones were considered as sterile. Counts were
taken in each cross and fertility/sterility was expressed in percentage.
| Table 1: | Dehydration of anthers gradually by using TBA dehydration series |
Anther Anatomy Study Through Sectioning
Paraffin sectioning was done as described by Johanson (1940). For this purpose,
anthers were collected in F1 sterile hybrid and F1 fertile
hybrid. The panicles were fixed in formalin (40% formaldehyde) : acetic acid
: alcohol (1:1:18) solution for 24 h at 4°C. The anthers were dissected
out from the spikelet and transferred to 50, 60 and 70%, ethanol each at one
hour intervals. The anthers were gradually dehydrated in Tertiary Butyl Alcohol
(TBA) series as shown in the Table 1.
After dehydration the anthers were given soaked in complete TBA twice at 6 h intervals. During the final change, a pinch of eosine was added to stain the specimen superficially so as to locate them inside the wax while embedding stage.
The specimen was transferred to TBA : liquid paraffin (1:1) for 2-3 h to a beaker containing three-fourth of solid wax (melting point 56-58°C). An additional quantity of TBA : liquid paraffine mixture was again added to the specimen and the beaker was kept at 60-65°C for 12 h. Two soakings were done with paraffine wax at 6 h intervals. The specimen was embedded in glycerin coated paper boat and the wax was rapidly cooled in ice water to get wax blocks which were stored at 4°C.
Sections of 10-12 μ thickness were cut and the ribbons were stretched out by placing them in warm water (40°C). These ribbons were cut into appropriate size and received on a micro slide coated with Haupt`s adhesive. The slides were flooded with 4% formalin and kept in warm plate (40°C) to get maximum stretching of ribbons. Then the excess fluid on the slide was drained and the slides were shade dried for 10-12 h in a dust free chamber.
The slides were passed through the following series:
| • |
Xylene-l, xylene-ll, xylene ethanol (1 : 1)-30 min each |
| • | Ethanol-100, 95, 70, 50, 30 and 10% and distilled water-10 min each |
| • | Ferrous ammonium sulphate (4%), 1% Erlich`s hematoxyline (ripened for 30 days) - 30 min each |
| • | Washed in running tap water (alkaline) for 10-15 min |
| • | Ethanol 10, 50, 70 and 100% - 5 min each |
| • | Xylene : ethanol (1:1), xylene-l, xylene-ll - 15 min each |
Then the slides were mounted in DPX mountant and left undisturbed for 24 h for examination. Sample were examined under a light microscope and photographed.
RESULTS AND DISCUSSION
Fertility Restoration Studies
Fertility restoration pattern of F1 hybrids developed from crosses
between male sterile lines possessing different CMS sources and a set of inbreds
have been used for the classification of CMS sources in pearl millet (Burton
and Athwal, 1967; Akenova and Chheda, 1981; Hanna, 1989; Rai and Hash, 1990).
Based on pollen fertility and seed set in the five lines and thirty inbreds
were classified as non restorers when there was less than 15%, while the others
were classified as restores. Such classification based on seed set has been
adopted by Hanna (1989).
The fertility restoration ability of thirty diverse inbred pollinators on different cytoplasmic sources were studied and the results are given in the Table 2. Among the one hundred and fifty hybrids studied, forty one hybrids were complete sterile and twenty four hybrids were fertile (Table 3). Among thirty inbreds, seven inbreds showed sterility with ICMA 88004A (PT 5232, PT 5259, PT 5426, PT 5440, PT 5446, PT 5450, PT 5455) and six testers restored the fertility (PT 5297, PT 5423, PT5427, PT 5433, PT 5447 and PT 5575). Out of thirty testers, twelve showed sterility with 81 A (PT 5130, PT 5164, PT 5173, PT 5200, PT 5232, PT 5446, PT 5450, PT 5451, PT 5455, PT 5627, PT 5656 and PT 5773) and only three testers restored the fertility in 81A. In case of 732A, 10 inbreds restored the fertility (PT 5172, PT 5181, PT 5423, PT 5433, PT 5446, PT5502, PT 5627, PT 5636, PT 5656 and PT 5809) and only one inbred showed sterility with 732A.
Among the thirty inbreds, six inbred showed sterility with ICMA 94111A (PT 5164, PT 5259, PT 5294, PT 5440, PT 5446 and PT 5773) and four inbreds restored the fertility (PT5173, PT 5181, PT 5232 and PT 5423). Fifteen inbreds showed sterility with PT 5054A (PT 5164, PT 5181, PT 5232, PT 5259, PT 5260, PT 5426, PT 5427, PT 5433, PT 5440, PT 5451, PT 5455, PT 5480, PT 5627, PT 5636 and PT 5773) and only one tester restored the fertility with PT 5054A. The testers PT 5164, PT 5232, PT5259, PT5440, PT 5446 and PT 5455 showed sterility with three male sterile lines out of five and the testers PT 5171, PT5181, PT 5297, PT 5427 and PT 5433 restored the fertility with two male sterile lines out of five male sterile line used. PT 5423 restored the fertility with ICMA 88004A, ICMA 94111A and 732A.
Anther Anatomy Studies Through Sectioning
Anther development and microsporogenesis in male sterile and fertile lines
were studied through sectioning. Developmental variation in the male sterile
and male fertile lines of pearl millet were studied in five developmental stages
viz., PMC, dyad, tetrad, soon after microspore release and before dehiscence
of anther (Fig. 1).
| Table 2: | The fertility restoration ability of thirty diverse inbred pollinators from different cytoplasmic sources |
| ss: Seed set percent, F: Fertile, S: Sterile, P: Partially | |
| Table 3: | Pollen sterility and seed set per cent of hybrids |
| ss: Seed set, ps: Pollen sterility | |
Microsporogenesis of cytoplasmic male sterile lines and male fertile lines was studied in order to identify the stage of breakdown of fertility during microsporogenesis which leads to male sterility in pearl millet. The major reason of the sterility was the abnormal behavior of the tapetal cells and bad timing of callose activity in anther. This result was conformed by the findings of Azhaguvel (1997) and Chhabra et al. (1997). In pollen mother cell, there is no variation in the wall layer in both male sterile line and male fertile lines. After the tetrad stage a very weak reaction and tapetum degeneration in the male fertile anthers were observed. The similar results were observed by Reddy and Reddi (1974) and Azhaguvel (1997). Microspores in sterile lines collapsed soon after their release from the tetrads. Similar observation has been recorded by Singh and Sharma (1963), Reddy and Reddi (1974) and Azhaguvel (1997).
In male fertile lines, tapetum thickness continued to increase and the central callose splitted along the sporocyte walls, following this, sporocytes migrated towards the inner tapetal wall carrying callose tips on their inner face. Callose breakdown occurred after the tetrad stage when young microspores were liberated. Microspores were oriented in a ring along the densely stained tapetum. Empty looking pollen grains slowly filled with starch granules and the tapetum degenerated. At this stage thickness of epidermis and endothecium was reduced. This result was conformed by the finding of Chhabra et al. (1997). Densely stained pollen grains observed in the male sterile lines in the contrast to the faintly stained irregular pollen grains in the male sterile lines. A similar result was observed by Azhaguvel (1997) and Chhabra et al. (1997).
| Fig. 1: | Transverse sections of fertile and sterile anthers representing microsporogenesis from young sporogenous stage to pollen maturity. A: Microspores were oriented in a ring along the densely stained tapetum; B: Empty looking pollen grains slowly filled with starch grainules; C and D: Well filled pollen grains with starch granules and fully developed exine and intine were seen at the anther maturity; E: In advanced stages of anther development cytoplasmic contraction of microspores and microspores started to shrinking; F and G: Microspores degenerated due to the inability of the fibrous tapetum to supply nutrition to developing microspores |
Anther Development and Microsporogenesis in Male Fertile Lines
The results showed that the intensity of protein reactions was similar in
all the wall layers. Prior to initiation of meiosis, young anthers contained
sporogeneous tissue surrounded by tapetum, followed by a middle layer, an endothecium
and the epidermis. Callose deposition started at the locule only after this
stage, became conspicuous at the onset of meiosis. Tapetum thickness continued
to increase and the central callose splitted along the sporocytes walls. Following
this, the sporocytes migrated towards the inner tapetal wall carrying callose
tips on their inner face. Callose breakdown occurred after the tetrad stage
when young microspores were liberated. Microspores were oriented in a ring along
the densely stained tapetum (Fig. 1A). Empty looking pollen
grains slowly filled with starch granules and the tapetum degenerated (Fig
1B). At this stage thickness of epidermis and endothecium was reduced. Well
filled pollen grains with starch granules and fully developed exine and intine
were seen at the anther maturity (Fig. 1C, D).
Anther Development and Microsporogenesis in Male Sterile Lines
At the dyad stage, the tapetum became tick and highly vacuolated. Young
microspores remained adhered to the tapetum, but later, microspores formed a
ring and migrated towards the locule interior. In advanced stages of anther
development cytoplasmic contraction of microspores and tapetal hypertrophy occurred
and microspores started to shrink (Fig. 1E). Ultimately,
microspores degenerated completely. Tapetal cytoplasm turned fibrous and microspores
degenerated due to the inability of the fibrous tapetum to supply nutrition
to developing microspores (Fig. 1F, G).
Tapetum cells increased in male fertile and sterile lines but declined in other wall layers. Soon after the release of microspore stage, all layers showed a very weak reaction and tapetum degenerated in the male fertile anther. The male sterile anthers were recorded very in high colour intensity in the tapetal layer and microspore collapsed immediately after separation from tetrad. Unlike the fertile line, in sterile lines, the tapetum at this stage persisted. Before dehiscence of anther stage, the male fertile lines showed that the pollen grains were densely stained.
CONCLUSION
In the present study, out of one hundred and fifty hybrids consisting of five male sterile lines and thirty inbred testers were studied, forty three hybrids expressed sterility and twenty four hybrids complete fertile. The hybrids expressed the sterility would be utilized for maintainer line development by backcrossing with the respective inbred lines and the inbred which restored the fertility of the male sterile line will be exploit for commercial hybrid vigour.
The two inbreds namely PT 5259 and PT 5773 recorded sterility with four male sterile lines out of five male sterile lines used. The former expressed sterility with ICMA 88994A, 81A, ICMA 94111A and PT 5054A, the later expressed sterility with the male sterile lines 81A, 732A, ICMA 94111A and PT 5054A. Based on the present investigation, the A line PT 5054A expressed sterility with fifteen hybrids out of inbreds studied and it was followed by 81A with fourteen inbreds. The 732A cytoplasm expressed sterility with only one inbred (PT5773) and all others are fertile and/or partial fertile and also it was registered complete fertility with most of the inbreds than other male sterile lines.
Based on these results obtained from microsporogenesis studies, it was found that, break down of fertility in the sterile anthers of pearl millet was post meiotic, in specific soon after the release of tetrad in the developmental stage. Microspore degenerated due to inability of the fibrous tapetum to supply nutrition to developing microspores. Tapetum cells increased in male fertile and sterile lines but declined in other wall layers. The male sterile anthers were recorded very in high colour intensity in the tapetal layer and microspore collapsed immediately after separation from tetrad. Unlike the fertile line, in sterile lines, the tapetum at this stage persisted. Before dehiscence of anther stage, the male fertile lines showed that the pollen grains were densely stained.