Genetic Studies

One of the most important landmarks in plant breeding history has been the successful utilization of Saccharum spontaneum in sugarcane improvement. In no other crop wild species has been so effectively utilized to meet human needs. This wild species S. spontaneum shows high levels of resistance against the whole gamut of biotic and abiotic stresses. Dr. C. A. Barber, the then Sugarcane Specialist at this Institute, struck upon the ingenious idea of using this wild species for sugarcane improvement. He crossed S. officinarum as female to S. spontaneum as male with the idea of infusing resistance against biotic and abiotic stresses in the cultivated sugarcane. The initial success with Co 205 as a popular variety in the subtropical belt created an explosion of further varietal development work that revolutionized sugarcane agriculture not only in India but also in the entire sugarcane growing countries of the world.

Apart from the ingenuity of Dr. Barber, there was another very important factor that made the breeding efforts successful at this Institute. Flowering and seed set under natural conditions is a very serious problem in sugarcane that hampers varietal development work the world-over. There appears to be a trend in sugarcane flowering given the specific location of a place in the globe. If sufficient growth of the crop is made available, sugarcane flowers throughout the year in the equatorial region, but is rendered completely sterile precluding any crop improvement activities in this region. As one moves away from the equatorial plane towards north or south, flowering period becomes more and more restricted, but the fertility level gets increased. At places situated around 11^o north and south latitude, flowering becomes restricted to 2-3 months in a year. At this latitude in the northern hemisphere, sugarcane flowers from October middle to January middle where as in the southern hemisphere it flowers from April middle to July middle. Fertility levels are the maximum in these regions and hence majority of the sugarcane breeding stations are situated here. Coimbatore is one such place and it has certain added advantages: It receives rains in both the monsoon seasons and is situated in the leeward side of the western ghats that provides ideal conditions of temperature, humidity and gentle winds for flowering and seed set. Thus flowering and seed set under natural conditions in the world are the best at Coimbatore.

In places falling away from 11^o latitude, flowering under natural conditions is extremely difficult to obtain. In such places appropriate artificial techniques are employed to make sugarcane flower.

Nobilization

The early Dutch workers called S. officinarum as the ‘noble cane’ due to its splendid appearance. The term ‘nobilization’ was first used by the Dutch sugarcane breeder Jeswiet in Java when referring to a crossing and back-crossing schedule by which progressive improvement of the hardy and disease resistant but otherwise inferior wild cane types is effected by crosses with the more attractive and sweeter noble canes (Stevenson, 1965). However, it should be noted that the back-crosses in such a programme are so termed in a general rather than a strict genetical sense, since the successive noble cane varieties may be (and in fact have usually been) different clones of S.officinarum, in order to bring in diversity.

Click here to view process of chromosome transmission during nobilization

The cytogenetical peculiarity in this nobilization process is that the hybrid progenies receive 2n gametes from S. officinarum and n gametes from S. spontaneum. This was established way back by Bremer (1923). In the reciprocal crosses however, only n + n transmission is obtained. Again when the hybrid is backcrossed to S. officinarum as the female, this process of 2n + n transmission is repeated. In further backcrosses 2n gametes may or may not function depending upon the specific clones of S. officinarum that might be used. This 2n + n transmission during nobilization is nature’s gift to sugarcane breeder since the entire genome of cultivated species is retained making it easy to eliminate most of the unwanted chromosomes of the wild species, while retaining the desirable ones. The term nobilization has since been expanded to include crossing of any wild species of Saccharum or its related genera to S.officinarum. Some times crossing of cultivars with S.spontaneum also is called nobilization which is better avoided.

Sugarcane Genepool

S. officinarum is the original sugarcane species. It is supposed to have originated in the Indonesian Archipelago. The species does not occur wild in nature but was grown and maintained for a long time by the natives of these islands. Later on it came to be cultivated in south India. There are two other cultivated species: S. barberi and S. sinense. These species are believed to be hybrid derivatives involving S. officinarum and wild species. S.barberi was grown in northern parts of India, while S. sinensewas cultivated in China. These two species are no more under cultivation with the advent of improved varieties that have been developed through complex hybridization. There are several other species and genera related to sugarcane.

Commercial cultivars, the products of S. officinarum, S. spontaneum, S. barberi and S. sinense, constitute the primary gene pool since potential cultivars can be fixed in the first sexual generation itself. The cultivated species S. officinarum, S. barberi and S. sinense can be considered as secondary gene pool since involvement of these species in varietal improvement would involve a few generations of breeding. S. spontaneum and S. robustum constitute the tertiary genepool since it takes a number of generations to eliminate the undesirable effects of these wild species during varietal development. The allied genera can be termed as distant gene pool and these have not been successfully utilised so far. Of these, the only cane forming species Erianthus arundinaceus is drawing a lot of attention of the breeders world wide especially because of its high biomass producing ability and resistance to biotic and abiotic stesses.

From breeding point of view it is sufficient to consider only S. officinarum, S.spontaneum, S. robustum and Erianthus arundinaceus since S. barberi and S. sinense are widely believed to be materials of introgression between S. officinarum and S.spontaneum (Brandes, 1958). Among the three wild species, utilisation of S. robustum has not been very successful so far, perhaps because it is genetically very much close to S.officinarum as revealed by investigations employing molecular techniques. Hence,the cultivated S. officinarum and the wild S. spontaneum and Erianthus arundinaceus, in addition to the vast array of commercial varieties, constitute the most important materials available to the breeder for sugarcane improvement.

The present day commercial varieties are the products of crossing, inter-crossing and back-crossing involving these two species in addition to the cultivated S. barberi and S.sinense. These cultivated varieties combine all the desirable attributes of S. officinarum and the resistance factors of S. spontaneum in addition to its ability to produce high biomass.

Mechanism of 2n transmission through egg cells

A number of hypotheses have been put forward to explain the mechanism of 2n + n transmission in sugarcane (Price, 1961). Among these are:

♦ Formation of unreduced egg cells

♦ Chromosome doubling through endo-duplication either at the dyad or tetrad stage

♦ Postmeiotic fusion of the two innermost megaspores

♦ Postmeiotic endomitosis in the egg cell

♦Incompatability of n + n gametes due to either selective fertilization or a combination of selective fertilization and parthenogenesis, i.e. differential survival of 2n+n and n+n zygotes

♦Failure of certain zygotic combinations due to faulty endosperm development (selective survival)

The exact mechanism of the formation of egg cells with two haploid sets of chromosomes is not unambiguously established. The possibility of unreduced egg cell formation can be precluded, since segregation of the maternal characters is observed among the hybrids from crosses between S. officinarum x S. spontaneum.The increase in chromosome number might occur by separation of chromatids of the S. officinarum chromosomes in the egg-nucleus either before or during fertilisation with a sperm nucleus of S.spontaneum. Or, chromosome doubling could occur in the chalazal megaspore by means of endoduplication, thus producing egg cells with n+n chromosomes, either at the dyad stage after the first meiotic division or at the tetrad stage after second division but before fertilisation. However, even this mechanism, while accounting for 2n+n zygotes, does not explain the transmission of the n chromosome number of S.officinarum on selfing or intraspecific crossing; 2n+n transmission occurs only after pollination and fertilisation following interspecific crossing. From this it could be inferred that S. officinarum produce both reduced (haploid) and unreduced egg cells. Two explanations of such a phenomenon would be either that 2n and n egg cells are selectively fertilised or that the chromosome doubling in the egg cell occurs at the time the egg cell is fertilised by the sperm nucleus of S.spontaneum.

Auto-syndetic pairing

In spite of presence of quite different genomes, there is no multivalent/univalent formation in any significant way in the hybrids as well as backcross generations of nobilization. Absence of this allo-syndetic pairing was thought to preclude chromosomal exchanges between genomes. Chromosome pairing in the immediate hybrids between S.officinarum x S. spontaneum is regular. Meiotic studies have shown that mostly bivalents and very few univalents are present at metapahse I (Bremer, 1961, Price, 1957). The univalents, when present, divided precociously, lagged at anaphase I, and were often included in the nuclei. Both the natural and synthetic hybrids between S.officinarum and S. spontaneum are male and female fertile. The average pollen fertility is about 75%, with a range of 40 to 100%. Complete male sterile progenies are also obtained very rarely and this must be due to genetic factors rather than meiotic behaviour.

Auto-syndetic pairing, if complete, precludes segmental interchange between genomes. Based on this, early sugarcane breeders came to the conclusion that there is no introgression of S.spontaneum chromosomes into S. officinarum genome. However, D’Hont et al. (1996) through comparative genomic DNA in situ hybridization studies, showed that in the cultivated clone, R570 (2n=107-115) about 10% of the chromosomes were identified as originating from S. spontaneum and about 10% were identified as recombinant chromosomes between the two species S. officinarum and S. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. This indicates that auto-syndetic pairing is not complete in the hybrid progenies and breeders might have selected unconsciously, segregants which might have resulted from rare allo-syndetic pairing.

Nobilization since1980s

It is essential to introduce greater diversity especially of S. spontaneum genome in imparting sustained resistance against abiotic stresses. Since the time Barber successfully made crosses between S. officinarum (Vellai) and S. spontaneum (Coimbatore form), the attempt was repeated several times upto the 60’s, using different clones in the process of evolving superior varieties. However, only 11 clones of S. officinarum and two clones of S.spontaneumas given below, have been utilised so far in the development of present day varieties:

S. officinarum: Ashy Mauritius, Badila, Banjermasin Hitam, Black Cheribon, Fidji,Green Sport, Kaluthai Boothan, Lahaina, Loethers, Striped Mauritius, Vellai.

S. spontaneum: Coimbatore form, Java form

Realising the need to introduce diverse genetic materials in the base population, intensive work in the last 15 years has resulted in the development of potential varieties that have in their ancestry, the following genetic base.

S. officinarum: 28 NG 51, 28 NG 93, 28 NG 210, 28 NG 221, 28 NG 224, 57 NG 78,57 NG 110, NG 77-63, NG 77-92, NG 77-99, NG 77-137, Uahi-e-pele.

S. spontaneum: SES 44A, SES 69, SES 87A, SES 90, SES 91, SES 93, SES 131, SES 198, SES 275, SES 515-7, SES 517 A, SES 538.

A number of promising materials developed from the above are under varietal evaluation in different areas and a few of them have been elevated to Co status.

Nobilization in E.arundinaceus

Apart from S.spontaneum, another important cane forming wild species, related to the genus Saccharum is Erianthus arundinaceus that can be nobilized. Its potentiality in contributing useful gene complexes for high fibre, high biomass, tolerance to drought and water logging, pest and disease resistance and multi-ratooning ability appears to be very attractive. However, there are two major obstacles that are to be overcome. The first is that S. officinarum, unlike in S.spontaneum nobilization, appears to transmit only n gametes instead of 2n when it is crossed to Erianthus arundinaceus. The second is that there is manifestation of sterility in the intergeneric hybrids, precluding further improvement through breeding.

A novel method of overcoming these twin difficulties has been thought of and is being implemented at this Institute. The idea is to use S.spontaneum as bridge species between S.officinarum and Erianthus arundinaceus. Towards this objective crosses have been made between Erianthus arundinaceus as female and S. spontaneum as male and vice versa. The presence of S. spontaneum genome in these intergeneric progenies, when used as males are expected to trigger 2n gametes formation in S.officinarum during the nobilization process and thus enable retention of the entire genome of S.officinarum.

Problems of using E. arundinaceus

The variability in E. arundinaceus collections is very much limited. Among the 70 and odd accessions that we have, only a few come to flowering. And these clones come to flowering early in the season, making synchronization with S. officinarum or varieties a problem. Collection and storage of pollen for a few weeks is an option being attempted to circumvent the problem. Creation of variability through selfing or open pollination may offer some scope for getting late flowering segregants.

E. arundinaceus as female: E.arundinaceus is extremely self fertile like S.spontaneum. Hence, when used as female, gives out a very large number of selfs.However, absence of a well-marked dewlap renders recognition of inter-generic hybrids an easy task – while all the selfs are characterized by absence of dewlap, the hybrids show presence of dewlap. In a week after germination, inter-gereric hybrids could thus be identified and raised separately.

E. arundinaceus as male: The time of anthesis is of E. arundinaceus very erratic and hence collection of pollen for hybridization is a difficult task. The anthers are tiny and it appears that decrease in humidity after sunrise that enables anthers of Saccharum species to dehisce, does not very much influence anthesis in E. arundinaeus. In order to circumvent this difficulty, the panicles could be covered with paper bags in the previous day itself so that all the pollen that might have shed during the night time could be collected in the morning at the time of hybridization. Androgenesis is a very common phenomenon when E. arundinaceus is used as male with Saccharum species as the female. Progenies arising out of this phenomenon could be easily identified at the seedling stage itself by the absence of dewlap and then eliminated.

Nobilization in S.spontaneum

Nobilization is very central to sugarcane breeding activities. Use of S.spontaneum in crossing with S. officinarium as female laid the foundation on which the sugarcane breeding activities have been built up. Cytogenetically, S.spontaneum has triggered 2n gamete functioning in S. officinarium enabling the hybrids to retain the entire genome of cultivated S. officinarium which is essential for further improvement. Agronomically, S.spontaneum has contributed desirable attributes like high biomass, resistance to pests and diseases and tolerance to adverse environmental factors. Thus, S.spontaneum has played a very effective role in sugarcane improvement. Erianthus arundinaceus, that is related to the genus Saccharum appears to be a very promising candidate for nobilization. Since sterility and absence of 2n + n chromosome transmission are the problems faced in nobilization E. arundinaceus, use of S. spontaneumas bridge species is being attempted and the preliminary results obtained are quite encouraging.

Out of several crosses attempted during 2001 crossing season in E. arundinaceus x S. spontaneum, 13 seedlings (confirmed hybrids) were obtained in two crosses as reported earlier. Out of these seedlings raised during 2002, four perished without forming stem. In the remaining nine seedlings, only three were healthy and came to flowering. The other six seedlings showed varying intensity of disorder in synthesising chlorophyll pigments. Because of this, they became very weak and did not come to flowering. The three progenies which were healthy, showed high pollen sterility. Thus, it is very clear that ‘ hybrid inviability ‘ and ‘hybrid sterility’ (as enunciated by Stebbins, 1951 in the context of seed propagated species) operate as reproductive barriers.

Chromosome number from root tip cells could be examined in four progenies in IK 76-92 x SES 286. Assuming n + n transmission the progenies should give a somatic number 62 (30 + 32). However, the 2n number was far less (50, 52, 54 and 58) indicating that there is en block elimination of chromosomes.

Although hybridity of these progenies was beyond doubt as ascertained by morphological features, molecular technique was employed to further confirm hybridity. Five of these nine surviving progenies were subjected to STMS (Sequence Tagged Microsatellites Sites) marker analysis and S. spontaneum specific markers were found, confirming hybridity. The three healthy progenies which showed 0 % pollen fertility as seedlings, gave certain degree of pollen fertility (5 to 15 %) after clonal propagation. These hybrid progenies were further utilised in making crosses. In the reciprocal group S. spontaneum x E. arundinaceus, several progenies were very vigorous and showed peculiar twining habit. The hybridity of these progenies is yet to be ascertained beyond doubt. Further investigation would reveal whether this new technique would bring in the much needed fertility to the intergeneric hybrids. Preliminary indications are that although the pollen fertility of the intergeneric hybrids between E. arundinaceus and S. spontaneum is very low (20 % or less), these clones when used as females in further hybridization, have given genuine hybrid progenies as indicated above.

References

♦ Brandes, E.W. 1958. Origin, classification and characteristics. In E. Artschwager and E.W. Brandes. Saccharum officinarum L. U.S. Dept. of Agric. Handbook. pp. 122 Bremer, G. 1923.A cytological investigation of some species and species hybrids within the genus Saccharum. Genetica, 5: 97-148.

♦ Bremer, G.1961. Problems in breeding and cytology of sugarcane. 1. A short history of sugarcane breeding – the original form of Saccharum. Euphytica, 10: 59-68.

♦ D’Hont, A, Grivet, L., Feldmann, P.,Rao, S., Berding, N., and Glaszmann, J.C. 1996. Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp.) by molecular cytogenetics. Molecular Genetics and Genomics. 250: 405-413.

♦ Piperidis, G., Christopher, M.J., Carroll, B.J., Berding, N. and D’Hont, A. 2000. Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus. Genome, 43(6): 1033-1037.

♦ Price, S. 1957. Cytological studies in Saccharum and allied genera. IV. Hybrids from S.officinarum (2n = 80) x S. spontaneum (2n + 96). J. Hered., 48: 141-145.

♦ Price, S. 1961. Cytological studies in Saccharum and allied genera. VII. Maternal chromosome transmission by S. officinarum in intra- and interspecific crosses. Bot.Gaz.,122, No.4.

♦ Stebbins,G.L. 1951. Variation and evolution in Plants.Columbia Univ. Press. New York, p.625.

♦ Stevenson,G.C. 1965.Genetics and Breeding of Sugarcane.Longmans, London, pp.284.