Embryo rescue: A tool to overcome incompatible interspecific hybridization in Gossypium Linn. A review

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1 Indian Journal of Biotechnology Vol 3, January 2004, pp Embryo rescue: A tool to overcome incompatible interspecific hybridization in Gossypium Linn. A review S S Mehetre * and A R Aher All India Coordinated Cotton Improvement Project, Mahatma Phule Krishi Vidyapeeth, Rahuri, Ahmednagar , India Received 10 September 2002; accepted 28 April 2003 Successful introgression of desirable characters from certain species could not be achieved because of different incompatibility reasons. Such incompatibility can be overcome by ovulo-embryo culture. From the literature reviewed so far, it appears that the technique involving excision of 10 to 15-day-old (after pollination) embryos and their subsequent culture on different combinations of BT and MS media was successful in obtaining difficult interspecific hybrids in Gossypium spp. BT and MS media supplemented with phytohormones, casein hydrolysate and IAA were more effective for culturing interspecific hybrid embryos. Other factors, like age and genotype of embryo; basal media, temperature and photoperiod during culturing; and hardening of plants are also important in determining the success of the interspecific embryo culture in Gossypium spp. Keywords: Gossypium spp., embryo rescue, interspecific Introduction Cotton is a leading fibre crop of the world belonging to the genus Gossypium, which consists of 49 identified and described species 1-3. Of these, 44 species are diploid (2n=2x=26) and fall into A to K genome and the remaining are allotetraploids (2n=2x=52) 4-6. These represent an aggregate geographic range that encompasses most tropical and subtropical regions of the world 7. Even though such a wide range of variability in wild species is available, certain crosses and many combinations are reported to be incompatible due to failure of fertilization between the species of Gossypium, viz. stocksii raimondii, stocksii gossypioides, sturtii aridum, sturtii gossypioides, thurberi armourianum, armourianum sturtii, armourianum raimondii, armourianum gossypioides, klotzschianum gossypioides, davidsonii herbaceum, davidsonii arboreum, aridum sturtii, aridum armourianum, aridum raimondii, raimondii stocksii, raimondii sturtii, raimondii thurberi, raimondii harknessii, *Author for correspondence: Tel: ; Fax: subhashmehetre@rediffmail.com Abbreviations: BT medium: Beasley & Ting medium (1971); 2,4-D: 2,4- diphenoxy acetic acis; EACA:E amino-n caproic acid (6, amino- N- hexanoic acid); GA: Gibberelic acid; IAA: Indole acetic acid; Kn: Kinetin; MS medium: Murashige & Skoog medium (1962) raimondii aridum, gossypioides stocksii, gossypioides sturtii, gossypioides armourianum, gossypioides klotzschianum, gossypioides davidsonii, and gossypioides x raimondii 8. In many of the interspecific hybrids, successful incorporation of wild species germplasm has been difficult because of the finely tuned genetic system controlling lint quality strength and texture 9, hybrid breakdown among interspecific hybrids 10,11, high negative correlation between fibre strength and high yield, and reduced chiasma frequencies 12,13. Ovuloembryo rescue for incompatible cross combinations has been resorted to secure gene(s) combinations that are not available within the limit of the species. In addition, it may be possible to obtain improvement in certain characters through transgressive breeding 14. Incompatibility caused by several pre- or postzygotic barriers is known to limit the production of hybrids between distantly related species 15. Blakeslee 16 and Stebbins 17 discussed various prefertilization barriers encountered in interspecific and intergeneric crosses resulting from: a) Failure of pollen germination, and b) Slow pollen tube growth. Slow pollen tube growth is one of the major crossability barriers restricting fertilization in wide crosses of cotton. For example, pollen of Hibiscus grandiflorus, even though germinated normally on the stigma of G. hirsutum, the pollen tube stopped growing at some point along the style 18. In cross between G. hirsutum G. herbaceum, pollen tube

2 30 INDIAN J BIOTECHNOL, JANUARY 2004 growth was delayed in the foreign tissue of the style, which later ruptured prematurely 19. Only 10 per cent pollen tubes reached the ovules in the cross between G. davidsonii G. harknessii. However, in case of G. klotzschianum G. harknessii, even though about 80 per cent of the pollen tubes reached the ovules of G. klotzschianum, their contents were not released in about 50% of the ovules. The major disturbances observed in fertilization were pro-embryonic and endosperms were formed in only in 9% ovules 20. Similarly, in cross between G. arboreum G. davidsonii, the percentage of ovules with pollen tubes in the interspecific cross and control was 20 and 80%, respectively 21. Post-fertilization barriers further hinder or retard the development of the zygote after fertilization and normal development of the seed. They include reproductive abnormalities in F 1 hybrids and their later generation progenies, such as elimination of chromosome, chromosome bridges and abnormalities, hybrid inviability, or weakness and hybrid sterility. The above abnormalities have been discussed by Mehetre et al 15 in considerable detail. Several workers have critically reviewed the compatibility relationships between various Gossypium spp Based on these, Sikka and Joshi 8 summarized certain broad conclusions regarding the interspecific cross compatibility relationships as follows: 1. Crosses between the old world species, excluding some of those involving G. sturtii, produce viable seeds. 2. With the exception of some of the crosses involving G. davidsonii and G. gossypioides, the crosses among the new world diploid species set viable seeds if fertilization is affected. 3. In crosses between the new world and the old world diploid species, excluding a few, fertilization is readily affected but many or all the seeds produced prove inviable. Incompatibility is apparently due to some post-fertilization disharmony. 4. Tetraploid species hybridize successfully with a majority of the diploid species excluding G. klotzschianum and G. davidsonii. 5. Interspecific crosses within tetraploid group are also readily made. Moreover, hybridization between two Gossypium species may fail at the following levels 25 : 1. The flower may drop soon after pollination without perceptible enlargement of the ovary, indicating that probably fertilization did not occur. 2. The ovary may enlarge and the young boll may remain on the plant several days to several weeks. 3. The bolls may mature and contain either abortive seeds or large empty seeds. In rare instances a hybrid embryo may mature, which may be smaller than a normal one. 4. After the development of an embryo, hybridization may fail. The seeds germinate but the seedlings die in the cotyledon stage. 5. At leaf stage and in some cases even at the flowering stage, death of the seedlings may take place. Several workers have attempted to develop techniques of embryo or ovulo culture as a means of overcoming incompatibility between the cotton species The most successful of them has been culturing of the ovules with the embryos enclosed. In a preliminary presentation, Pundir obtained small hybrid seedlings with A 2 cytoplasm from cultured ovules but presented few details of his procedure 31. Success in culturing AD ovules was first realized by Stewart and Hsu 33. They obtained a 50-60% yield of selfed embryos that could then be sub-cultured to establish the seedlings. They extended the technique of in ovulo embryo culture to cultured hybrid embryos form all possible crosses between the tetraploids (AD 1 AD 2 ) and the Asiatic diploids (A 1 A 2 ). However, they could not be able to obtain triploid (A 1 AD 2 ) mature hybrid plants. Techniques devised to overcome barriers between two incompatible species for successful production of viable hybrids are: 1) Bridging species technique, 2) Use of exogenous growth substances and immunosuppressants (GA, IAA, Kn, EACA, Chloramphenicol, aeriflavin etc.), 3) Pollen recognition, 4) Embryo rescue, 5) In vitro or in vivo embryo culture, 6) In vitro fertilization and ovule culture, 7) Mixed pollen technique, 8) Use of irradiation, 9) Use of stimulants, 10) Use of different pollination techniques, and 11) Grafting. Of these techniques, embryo rescue was found to be the most successful to obtain difficult or incompatible crosses of Gossypium species 15. Embryo culture may be defined as the process in which embryos of different developmental stages formed within the female gametophyte, through the normal sexual processes, is separated from the

3 MEHETRE & AHER: EMBRYO RESCUE IN INTERSPECIFIC HYBRIDIZATION 31 maternal tissues and cultured in vitro under aseptic conditions in a medium of known chemical composition. Initially, it is mainly employed in crosses between Gossypium species of hirsutum arboreum. G. hirsutum is tetraploid and susceptible to pests and drought, whereas cultivated desi, G. arboreum is tolerant to sucking pests as well as drought. Hence, introgression of these genes from G. arboreum to G. hirsutum cotton is worth attempting. As discussed earlier, there has been incompatibility in many crosses for disharmony between species concerned. Therefore, excision of hybrid embryos and their in vitro culture is a practical solution to overcome cross incompatibility problems. Further advances in embryo culture have opened up new vistas in obtaining plants from inviable hybrids. Techniques of Embryo Culture For excision of embryos and their transfer to nutrient medium, aseptic procedures are necessary. Since the embryos are lodged in a sterile environment of the ovule, surface sterilization of the embryos as such is not necessary. Instead, entire ovule, seed, or capsule containing ovules is sterilized and the embryos are freed aseptically from surrounding tissues. Isolation procedure begins with soaking the source in water for a few hours to a few days, so that the embryos can be removed with ease. Ovules are generally surface sterilized before soaking. They are then split open and the embryos are transferred to the nutrient medium. The most important aspect of embryo culture is the selection of the medium necessary to sustain its continued growth. Mature embryos are generally grown in an inorganic salt medium supplemented with a carbon energy source, such as sucrose. Relatively small pro-embryos, in addition, also require different combinations of vitamins, amino acids, growth hormones and natural endosperm extracts, like coconut milk. In vitro grown plantlets from embryos are then removed from the original medium and nurtured in sterilized soil or vermiculite and further grown to maturity in the greenhouse. Cytological and morphological characterisation of mature hybrid plants is done for the confirmation of hybridity. Historical Perspective Embryo was the first plant organ to be successfully cultured in vitro on artificial medium. For the next 50 years, together as a means of studying various aspects of embryology, the use of in vitro culture to rescue hybrid embryos remained a major application for a wide range of plant genera and families. The first report on successful in vitro embryo culture was by Hanning in 1904, who grew nearly mature embryos of Raphanus and Cochlearia on a mineral salts medium with sugars, amino acids and plant extracts 34. Hanning also described the problem of precocious germination of small immature embryos to produce weak and often inviable plantlets, a problem subsequently encountered by many other workers using immature embryos. Laibach was the first to successfully culture interspecific hybrid embryos 35. Using 15% glucose on a cotton wool substrate he was able to culture Linum hybrids from crosses that normally resulted in aborted seeds. He suggested that abortion in vivo might be due to lack of normal activity of the endosperm. The successful rescue of Linum hybrid embryos by Laibach elicited interest in embryo culture as a tool in plant breeding. Later, Tukey described embryo culture techniques in relation to fruit development for a number of fruit tree genera Beasley and Ting 39 developed a high salt BT medium for cotton that gave good ovule growth when supplemented with phytohormones (BTP); however, the report was only concerned with the development of the ovules and fibres for two weeks. Stewart and Hsu noted that supplementation of BTP medium with mm ammonium ions supported ovule growth and germination of cotton embryos 32. However, the size of the embryos remained small with reduced cotyledons even after 10 weeks of incubation without extra NH + 4. Later, others conducted extensive fertilization and embryological studies of G. hirsutum G. arboreum cross and their reciprocal crosses 31,40,41. Development of hybrid seedlings from crosses of G. hirsutum cv. Hencock, G. barbadense cv. Pima, G. herbaceum cv. Jaydhar and G. arboreum cv. Nan King have been reported 32. The cultured G. hirsutum ovules gave high yield of hybrid embryos without phytohormones. Gill and Bajaj used the ovulo-embryo culture technique for the development of complete plants in G. arboreum G. anomalum cross and the germination of seeds produced in other crosses, such as G. arboreum G. hirsutum and their reciprocals 42. Abortion of embryo at different stages of development has been a characteristic feature of wide crosses. Embryo rescue, however, was successfully been employed to produce hybrids involving interspecific and intergeneric crosses. Pollination of

4 32 INDIAN J BIOTECHNOL, JANUARY 2004 wild diploid Gossypium spp. (3) by cultivated spp. (4), followed by embryo rescue resulted in viable plants from G. trilobum G. hirsutum, G. stocksii G. hirsutum, G. stocksii G. arboreum and G. somalense G. arboreum crosses, where first species was used as female parent. Results indicated that the success of fertilization and embryo development was strongly influenced by the paternal species used in the cross and the degree of hybrid embryo development may be a more important factor than age or size at the time of embryo rescue. Immature embryos, rescued prior to 15 days post anthesis, failed to show further development or underwent precocious germination. The optimum time for embryo rescue and recovery occurred between 15 and 25 days after anthesis 42. However, Thengane et al excised 8 to 10-day-old ovules of G. hirsutum G. arboreum and cultured them in modified BT medium 44. Pentaploid (2n = 65) BC l plants, obtained from a cross between G. sturtianum (2n = 26 genome C l ) and G. hirsutum (2n = 52, AD 1 ), were pollinated in the field by G. hirsutum. As a result, 14 mature BC 2 plants with partial female fertility were produced by culture of fertilized ovules and embryo rescue. One BC 2 plant had no gossypol glands while in culture but developed them fully after the emergence of first true leaf. Subsequently, 8 BC 3 (2n = 55 or 57) and 125 BC 4 plants were derived from this plant, where seeds of five BC 4 plants were completely glandless 45. Hybrid plants from G. hirsutum G. harknessii, G. hirsutum G. raimondii and G. hirsutum G. armourianum crosses were successfully obtained by embryo rescue 46. Girhotra et al also obtained hybrids from three interspecific crosses, viz. G. arboreum G. thurberi, G. arboreum G. stocksii and G. arboreum G. anomalum, via embryo rescue technique 47. Crosses between G. hirsutum G. sturtianum were performed using G. raimondii or G. thurberi as bridging species. Recurrent backcrossing to G. hirsutum further as pollinator and selfing of second backcross (BC) progenies resulted in seeds that were rescued by in vitro culture 48. Post-fertilization incompatibility has also been shown by some of the cross combinations. Bolls formed in such situations showed a non-parallel development of embryo, seed coat and capsule, which was indicative of a fundamental disharmony between the hybrid zygote and the surrounding tissue. The physiological process responsible for the development of the different parts of the boll was interrupted at different thresholds, so that the development of all parts did not cease simultaneously resulting into non-parallel development 25. In cases of seed failure, e.g. mature bolls containing well-developed seed coats without embryos, it was suggested that once certain thresholds were crossed, the processes involved might continue more or less autonomously, regardless of the subsequent fate of the tissue that produced the initial stimulus. The precise determination of why and how particular crosses in this category failed must await histological studies. The failure of endosperm leading to the death of the embryo has been observed in the cross between G. arboreum G. davidsonii 49. Studies on the embryological development in cross G. hirsutum G. arboreum showed that hybrid endosperm and maternal tissue developed at a vigorous rate and in a fairly normal manner, resulting in large embryo less seeds, only when either the egg cell was not fertilized or the zygote failed to divide. In other cases, however, usually both the embryo and the endosperm started developing but soon get aborted. This abortion was considered probably due to a physiological imbalance between the two hybrid tissues, or more likely due to a degenerating effect of the hybrid embryo upon endosperm development 40. In crosses involving the old world, the new world and synthetic hexaploid cottons, the endosperm was the first tissue to show abnormalities 50. In crosses between females having low chromosome number and males having high chromosome number, the endosperm showed rapid initial growth but degenerated 10 to 20 days after pollination without developing cell walls. The greater the difference in chromosome number, the more rapid was the degeneration. The embryo often reached onefifth the normal size but did not differentiate properly. However, the maternal tissue developed normally. In other crosses (except in hexaploid tetraploid), the embryos grew normally for 10 days, whereas endosperm formed cell walls prematurely after five days. The maternal tissue stopped growth immediately after the endosperm 40. In such cases where normal growth and nutrition of the hybrid embryo is not possible in vivo, the embryos can be excised and nursed in vitro. Stephens and Cassidy attempted to nurse an arboreum davidsonii hybrid in this way but failed to obtain a viable plant 49. This may be due to a general genotypic disharmony between the species concerned 22,51. However, where there is no additional problem of genotypic

5 MEHETRE & AHER: EMBRYO RESCUE IN INTERSPECIFIC HYBRIDIZATION 33 differences, embryo culture has been successful. The hybrids between G. hirsutum G. barbadense or G. arboreum and G. herbaceum G. anomalum can be obtained by excising and artificially culturing hybrid embryos 52. Various combinations of auxins were required in the basal culture medium of BT to ensure the production of adequate numbers of embryos from fertilized ovules of Gossypium spp. except G. hirsutum, which required no growth regulator 33. It has been considered that the technique may be used to hybridize cultivated cotton and wild Gossypium spp. In embryo culture tests of interspecific crosses, 40 to 45-days-old embryos had the best average survival rate (80%) and White s was found to be the best culture medium 53. Self-pollinated and cross-fertilized embryos (2 and 15 days after pollination) from G. arboreum cv. G-27, G. herbaceum cv. SM-132, G-27 SM-132, and G-27 G. anomalum (a wild diploid) were cultured on various media. The 2-day-old embryos gave good callus growth on B5 medium supplemented with 2,4-D (2 mg/l), and 15-day-old embryos produced vigorous seedlings on MS medium supplemented with casein hydrolysate (250 mg/l). The 15-day-old embryos of G-27, SM-132 and their interspecific hybrid developed seedlings on MS medium supplemented with IAA (2 ppm) and Kn (0.5 ppm), which was found out to be the best medium for embryo culture 54. Mirza and Shaikh reported seedlings from embryo culture of 10 interspecific crosses 55. They successfully obtained matured plants of the crosses G. hirsutum G. herbaceum, G. hirsutum G. arboreum and G. hirsutum G. stocksii by embryo rescue. Embryos from G. arboreum G. stocksii, G. arboreum G. anomalum and G. herbaceum G. stocksii crosses when cultured 15 days after pollination and incubated in the dark for days before being exposed to light, the resulting plants were vigorous 56. Embryos of G. arboreum G. anomalum grew rapidly but more embryos of G. arboreum G. stocksii produced seedlings. In general, the characters of hybrid plants, subsequently grown in the field, were intermediate between those of the parents. However, the hybrids of G. arboreum G. anomalum were almost twice the size of the parents, and hybrids of G. arboreum G. anomalum were almost twice as tall as either parent and bore more flowers 56. Excised embryos from various crosses developed best on MS medium supplemented with IAA (1.5), Kn (0.5) and casein hydrolysate (250 mg/l). Germination and number of plantlets obtained ranged from 48% for Gossypium herbaceum G. stockssii to 71% for G. arboreum G. stocksii. Germination occurred within days of culturing. Those from diploid diploid crosses (G. arboreum G. anomalum) grew best on medium containing Kn (0.5 mg/l) and germinated in days of culturing, while those from tetraploid diploid crosses (G. hirsutum G. arboreum) grew best on medium containing Kn (0.2 mg/l) and germinated in days 42. In vivo or in vitro embryo culture is simply a modification of the embryo rescue technique as explained above. Plants from excised ovules of G. arboreum G. hirsutum 3 days after pollination and hybrid plants with a triploid chromosome number (2n = 3x = 39) were obtained using MS medium 57. Dhumale et al harvested developing bolls from crosses between G. arboreum G. hirsutum 15 days after pollination 58. They obtained hybrid plants by culturing aseptically dissected embryos initially on BT 1 medium and subsequently on BT 2 and BT 3 media. The embryo culture technique (with White s medium) has been used to overcome inviability in G. davidsonii G. sturtii hybrid 59,52. Embryo culture has made it possible to hybridize G. arboreum and G. armourianum. Liang et al successfully regenerated embryo from G. hirsutum G. sturtianum cross 60. Embryos of the cross between G. arboreum G. hirsutum were cultured 12, 15, 20 and 25 days after pollination 61. Mirza and Shaikh obtained mature plants from G. hirsutum G. stocksii cross by germinating embryos through ovule culture on BT medium 55. In vitro Fertilization and Ovule Culture In vitro fertilization, followed by culturing fertilized ovules to maturity, is an important technique employed to overcome the barriers inhibiting pollen tube growth and very early stage embryo abortion. It is a promising approach and a viable alternative to parasexual or somatic cell hybridization. Stewart and Hsu described a technique involving in vitro culture after in situ cross pollination and fertilization, which made hybridization possible between diploid old world G. herbaceum and G. arboreum (2n = 26) with

6 34 INDIAN J BIOTECHNOL, JANUARY 2004 normally incompatible tetraploid new world species, G. hirsutum and G. barbadense (2n = 52) 33. Factors Affecting Embryo Culture 1. Age The extent of embryo growth in cotton is related to the age of the ovule at the time of culture. Beasley et al observed that isolated fertilized cotton ovules could be maintained in culture to the point of germination 30. They reported complete embryo development from ovules cultured at pro-embryo stage (2 days post anthesis). Eid et al reported the growth of 5-day-old cotton ovules in culture up to the early dicotyledon stage. On the other hand, culture of 10 to 15-day-old ovules were reported to produce normal seedlings 26. They concluded that the variation in embryo development was associated with the developmental stage of endosperm present in the ovule. 2. Temperature Temperature is an important factor affecting the viability of the hybrid embryos. Viability of the cotton hybrid embryos was temperature dependent 44. At higher ambient temperature (40-42 C) abscission of bolls occurred within 4-5 days after pollination. Ovules from such abscised bolls did not develop in culture Basal media Different basal media, viz. White s 63, MS 64 and BT 39, were used for cotton ovule culture. However, the growth of the ovules was poor on White s and MS media as compared to BT medium. In a study, BT medium was reported to support in vitro growth and germination of self fertilized G. hirsutum embryos only if supplemented with phytohormones. It was noticed that nitrate reductase activity in the cultured ovules was inadequate to supply the requisite reduced nitrogen to the ovule integument and endospermembryo complex; hence, an external supply of NH 4 + ions was needed. Further, the level and form of nitrogen in the culture medium was found critical for in ovulo embryo growth in cotton; NH + 4 in the form of NH 4 NO 3 was found promoting better growth and germination of G. hirsutum embryos 32. Stewart and Hsu reported increase in the yield of hybrid cotton embryos with 15 mm NH 4 Cl 33. Prevention of precocious germination by supplementation of higher sucrose levels in the medium can be done. Thengane et al used 2 to 12% sucrose in the medium and observed that with the increase of sucrose concentrations up to 5%, there was a concomitant increase in the percentage of germination 44. Beyond 5% sucrose, the ovules enlarged and burst releasing a mucilaginous fluid, ultimately giving rise to a heavy growth of soft callus. 4. Genotype Embryo culture response depends upon the genotype of the parent from which embryo has been taken Hardening Apart from embryo culture, the success of the whole process lies in the survival of the plantlets during hardening. Six hardening treatments were compared and it was found out that maximum survival (90%) was observed by transferring plantlets to sterile distilled water for 6 hrs, then to trays covered with polythene sheet and keeping in open; followed by transferring plantlets to sterile distilled water for 6hrs, then to trays kept in mist chamber (80%) 61. Conclusion Ovule culture is an important tool for overcoming incompatibility amongst parents and successfully obtaining hybrids from difficult, distant cross combinations. Thus, from wild Gossypium species to cultigens, introgression of desired genes is made possible. Since most commercial cotton production involves tetraploid G. hirsutum, considerable interest has been generated for broadening the available genetic base. Hybrids between tetraploid and diploid Gossypium species are considered useful for introducing improved agronomic and quality traits into commercial cotton. Cotton breeders have long been interested in producing hybrids between G. arboreum and G. hirsutum to combine several desirable characters. However, such hybrids are impossible to obtain in situ. The reasons being endosperm and embryo abortion, and possibly other incompatibility factors apart from the failure of pollen tube to reach the ovule. In the beginning, several attempts have been made to excise and grow young embryos of cotton but they all met with limited success. The difficulties were attributed to sensitivity of the embryos to physical conditions and also to their complex nutrient requirements. As an alternative, therefore, ovule culture has been used with some degree of success. For cotton, high salt BT medium gave good ovule growth when supplemented with phytohormones (BTP). Further, supplementation of BTP medium with

7 MEHETRE & AHER: EMBRYO RESCUE IN INTERSPECIFIC HYBRIDIZATION mm ammonium ions supported the ovule growth and germination of cotton embryos. Without extra NH + 4, the size of embryos remained small with reduced cotyledons even after 10 weeks of incubation. Extensive fertilization and embryological studies of G. hirsutum G. arboreum cross and their reciprocal crosses demonstrated that both endospermic and embryonic abortions occurred in the crosses. However, hybrid seedlings of cotton were produced through culturing ovules. Complete plants from G. arboreum G. anomalum and G. arboreum G. hirsutum crosses along with their reciprocals were achieved through in vitro or in ovulo embryo cultures. Since most commercial cotton production involves tetraploid G. hirsutum, considerable interest has been generated for broadening the available genetic base. 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