CryoLetters 24, 119-124 (2003) Ó CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK
POLLEN FROM Glycine SPECIES SURVIVE CRYOGENIC EXPOSURE R.K. Tyagi* and T. Hymowitz Department of Crop Sciences, University of Illinois, 1102 South Goodwin Avenue, Urbana IL 61801, USA *
Corresponding author, Present address: National Bureau of Plant Genetic Resources, New Delhi-110 012, India . E-mail:
[email protected] Abstract Pollen of 12 genotypes of the annual soybean and its wild perennial relatives were stored without pre-desiccation at low temperatures (-20ºC and -196ºC) and tested for their viability in vitro. The influence of cryopreserved pollen on pod set and seed production was also investigated. Cryopreserved pollen of all the genotypes showed germination in vitro. Pollen of annual soybean stored at -20ºC retained their viability for 4 months, however, pollen of its wild perennial relatives at same storage conditions failed to germinate in vitro. Flowers pollinated with cryopreserved pollen had similar pod set and number of seeds/pod as those pollinated with fresh pollen. Results of this study suggest that cryopreservation of pollen can be used successfully for soybean breeding, and also offers the possibility of conserving the haploid gene pool of soybean and wild perennial species in a cryobank facility. Keywords: Cryopreservation, hybridization, germplasm conservation, Glycine spp., soybean INTRODUCTION Maintaining pollen viability and fertility for a long period is important in plant breeding in order to cross-pollinate the plants that flower asynchronously or are grown at different times and locations. In addition, pollen grains are stored for exchanging germplasm, preserving nuclear genes of germplasm, for basic studies concerned with gene expression, transformation and in vitro fertilization (21). A number of studies have been reported for pollen storage of various crops under various storage conditions (21, 22). Soybean, Glycine max (L.) Merr., is the most important grain legume in the world in terms of production and international trade (3).Wild perennial species of Glycine are rich source of useful genes for resistance to major pest and tolerance to abiotic stresses (5, 8, 9, 10, 14, 18). Hymowitz and his co-workers have been working for many years on wide hybridization of soybean with wild species having the objectives to introduce the alien useful genes into soybean (19). The flowering in soybean and its wild perennials is asynchronous and poses difficulty in pollination due to non-availabilty of sufficient pollen from the parents. Therefore, development of storage methods of pollen of these species will enable to continue the pollination without having to synchronize the flowering of both the parents and also have pollen available at different growing locations.
We found no applicable data on low temperature storage of pollen of Glycine species, pollen viability and fertility. In this study pollen of 12 genotypes of three Glycine species were stored under low temperature and results are reported in the present paper. MATERIALS AND METHODS Plant material Experiments were conducted with pollen of one accession of Glycine canescens – PI 440932 (2n = 40), seven cultivars of G. max (2n = 40) – cvs. Altona, Ina, Jack, Kunitz, Rend, Spencer, Williams 82 and four accessions of G. tomentella – PI 440998 (2n = 38), PI 440930 (2n = 40), PI 483224 (2n = 78), PI 573073 (2n = 80). The plants of these genotypes were grown in the greenhouse at the Department of Crop Sciences, University of Illinois, Urbana, under 16 h photoperiod cycle at 25 + 2ºC with 1197 mol m-2s-1 photosynthetic active radiation. The plants were grown in clay pots containing soil:perlite:torpedo sand (1:1:1). Pollen collection To study the feasibility of pollen germination several pilot experiments were conducted with the pollen of G. tomentella, PI 440930. Pollen were collected from 15 to 20 closed, halfopen and completely open flowers in the morning (8:30 h – 10:30 h) and afternoon (2:00 h – 3:00 h) and studied for pollen germination. Dictated by the results of pilot experiments, pollen grains were collected in morning (8:30 h – 10:30 h) from fresh completely open flowers and used to study the pollen germination in 12 genotypes. The freshly dehisced anthers were placed on a glass microslide and gently crushed with the help of needle to extract out the pollen mass. For a given genotype, pollen from 50 to 70 fresh and completely open flowers from 2 or 3 plants were mixed thoroughly to prepare aliquots for experimentation. Pollen was used within 2 h for experimentation. Low temperature storage The pollen samples from the above were carefully transferred into cryovials (1 ml, Fisherbrand, Pittsburgh, PA). Aliquots of pollen were stored for four months at -20ºC in a freezer and for 7 days at -196ºC in a metal-cased cylindrical cryoflask containing liquid nitrogen (LN2). The complete emersion of the sample was ensured in LN 2 by maintaining the LN2 level well above the sample through frequent filling of the cryoflask. Pollen germination Pollen germination was studied using the sitting drop culture method (15). As a pilot experiment, pollen grains of G. tomentella, PI 440930, were sprinkled onto a drop of boric acid (15 and 30 mg/l) and sucrose (5% and 10%) on microslide and allowed to germinate for 2 h at room temperature (22 + 2ºC) in a closed Petri dish lined by moist filter paper to maintain high humidity (70%-80%). Dictated by the results of above experiments, only one medium (30 mg/l boric acid and 10% sucrose) was used to study the pollen germination with the same set of culture conditions in all the tested genotypes. For each genotype a minimum of 2 cultures were raised on 3 different occasions. The cultures were terminated by adding a drop of 1% acetocarmine. From each culture up to three microscopic fields and 535-720 pollen were scored for germination. A pollen grain was considered as germinated when the length of pollen tube exceeded the diameter of the pollen grain. The experiments were conducted in completely randomized block design. Analysis of variance and Duncan’s Multiple Range Test were computed using standard software applicable to randomized complete block design.
Fertility assessment of cryopreserved pollen Fertility of the cryopreserved pollen of G. canescens, PI 440932; G. max cvs. Spencer and Williams 82; and G. tomentella, PI 573073 were assessed by pollinating the flowers of same genotype with the cryostored pollen as well as fresh pollen (control). Depending upon the availability of pollen and flowers, some 24 to 55 flowers were pollinated. Two or three plants of each genotype were used as females. Stigmas were thoroughly coated with pollen. Pod and seed set were recorded after maturity. RESULTS AND DISCUSSION Pollen germination Some 85% pollen germination on boric acid (30 mg/l) and sucrose (10%) was recorded for the pollen collected from completely fresh open flowers in G. tomentella, PI 440930 whereas in half-open and closed flowers, pollen germination was 60.4% and 43.8%, respectively, on same medium. No significant effect of natural light was observed as pollen collected during bright sunny days and cloudy days showed about 87% and 82% pollen germination, respectively, in G. tomentella, PI 440930. This may be attributed to the fact that plants were grown under controlled light conditions in the greenhouse. Therefore, pollen was collected from fresh and completely open flowers to study the in vitro pollen germination in fresh as well as stored pollen of 12 genotypes using boric acid (30 mg/l) and sucrose (10%) medium. Data on pollen germination percentage of fresh and stored pollen are presented in Table 1. The statistical analysis showed that the differences in pollen germination among the genotypes (F = 190.2, df = 11, P £ 0.01), among the treatments (F = 176.4, df = 2, P £ 0.01), and interaction between the genotypes and treatments (F = 19.4, df = 22, P £ 0.01) are highly significant. All the genotypes showed pollen germination in fresh as well as cryostored pollen on boric acid (30 mg/l) and sucrose (10%). The high percentage of pollen germination in partially sterile plants of soybean on 7.5 mg/l or 30 mg/l boric acid and 5% or 10% sucrose has already been reported (12). Considering together all the genotypes tested, pollen germination ranged from 24.4% to 92.9% in fresh pollen and from 17.2% to 77.8% in cryostored pollen. Pollen germination of G. max was observed as 65.1% before storage and 24% after storage in liquid air (-192ºC) (4). In present study, pollen of all the seven genotypes of G. max maintained good viability after storage for 4 months at -20ºC whereas pollen of wild perennials was not found viable at -20ºC. We suggest that further investigations are needed to study the effects of interaction of water content and temperature for storage of pollen particularly in case of wild perennials of Glycine species. Soybean pollen grains are binucleate and are generally desiccation-tolerant (4). Such pollen, with a moisture level below that which constitutes freezable water, usually has a viability after cryopreservation approximating that of uncooled samples. There are, however, instances where the viability of low temperature stored samples is considerably lower than that of uncooled sample. Reasons for this decreased viability are not apparent (20). Experimental evidences suggest that optimum water content for storage of pollen is an important factor for maintaining the viability of pollen under low temperature storage (1, 2, 23). Pre-desiccated cryostored pollen of rose (13) and yam (11) did not show any decrease in pollen germination. In our studies, no efforts were made to decrease the moisture content of pollen for the sake of practical convenience in handling the pollen for storage and pollination. Though, most of the genotypes showed good percentage of pollen germination after storage in LN2 without pre-desiccation.
Table 1. In vitro pollen germination (%) in Glycine species Species, genotype Fresh pollen G. canescens PI 440932 (2n = 40) Glycine max (2n = 40) Altona Ina Jack Kunitz Rend Spencer Williams 82 G. tomentella PI 440998 (2n = 38) PI 440930 (2n = 40) PI 483224 (2n = 78) PI 573073 (2n = 80)
Pollen germination (%) Stored at -20ºC Stored at -196ºC
88.7a,b
0
42.7j
84.4 a,b,c,d 76.3 d,e 74.3e,f 77.7c,d,e 92.9 a 86.9 a,b,c 78.7 c,d,e
60.9g,h,i 62.3g,h 59.4g,h,i 52.5i 61.8g,h,i 63.6g,h 66.6f,g
63.9g,h 66.8f,g 66.5f,g 56.3h,i 82.7 b,c,d,e 76.1 d,e 62.2 g,h
32.1k,l 77.2d,e 38.0j,k 24.4l,m
0 0 0 0
24.1 l,m 52.6 i 29.4 k,l 17.2 m
Values superscripted with different alphabets are significantly different at the 5% level using Duncan’s multiple range test
Fertility assessment of cryopreserved pollen Cryopreserved pollen was able to effect normal fertilization. The percentage of pod setting effected by cryopreserved pollen ranged from 23%-29%, which is comparable with the pod setting effected by pollination with fresh pollen i.e. 18%-30% (Table 2). The fertilized flowers that did not set the pods, eventually aborted within 3-4 days of pollination. In G. canescens, the average number of seeds/pod effected from cryostored pollen decreased from 7.2 to 3.2 but the converse was true for G. tomentella. However, both the cultivars of G. max remained unaffected. Pollen fertility could be retained in all the crosses, as indexed by fertilization and seed formation. The successful pollination leading to seed production depends on the ability of pollen to deliver successfully the male gamete to the embryo sac (17). In our studies, no apparent adverse effects of cryopreservation on pollen tube length in vitro were observed. Therefore, pollen tubes were capable of delivering male gametes to effect fertilization as evidenced by normal seed set after pollination with cryostored pollen. In the present investigation, viability and fertility could be retained in cryopreserved pollen for seven days. Pollen stored in LN2 should theoretically retain viability beyond this duration since at this temperature all biological activities are considered to be in a suspended animation (24). No significant difference was observed in pollen germination between three cultivars of rose pollen cryopreserved for 24 h and one year (13). The ability of cryopreserved pollen to germinate in vitro and effect the fertilization to set pods and seed on same parents, shows the feasibility of cryopreservation of pollen of soybean and its wild perennials. However, the cryopreserved pollen of wild perennials, generally showed low pollen germination percentages, but it did not affect the pod and seed set. Cryopreserved pollen of G. tomentella,
PI 573073, with pollen germination as low as 17%, was able to effect fertilization as successfully well as any other genotypes that had higher pollen germination. Seed formation may occur even if the in vitro pollen germination indicated low viability (20). Use of stored pollen for wide crosses in wheat haploid breeding has been very successful (6). High seed percentage with stored pollen used in vivo was also reported (7). It is quite common that stored pollen that shows low viability in vitro can induce satisfactory pod and seed set; pollen which does not show germination in vitro after storage cannot be adjudged non-viable and, conversely, pollen germinating well in vitro may not effect satisfactory seed set (15). Table 2. Seed set effected by pollinations using fresh and cryopreserved pollen Parent
Fresh pollen Number of Number Number flowers of pod of pollinated set (%)* seeds/ pod
Cryopreserved pollen Number of Number Number flowers of pod of seeds/ pollinated set (%)* pod
G. canescens PI 440932 42 12 (29) 7.2 45 G. max Spencer 45 8 (18) 2.5 48 Williams 82 30 9 (30) 2.0 24 G. tomentella PI 573073 55 15 (27) 4.0 52 * Values in parentheses are percentages for pod set rounded to the
11 (24)
3.2
12 (25) 7 (29)
2.3 2.0
12 (23) 6.1 nearest integer
We have successfully cryopreserved the pollen of 12 genoypes of three Glycine species. As noted in other crop species (22), cryopreservation is likely to be feasible for other species of Glycine also. Thus, storage of pollen should increase efficiency in using resources for wide hybridization in Glycine species and offers the possibility of conserving their haploid gene pool in cryobank. Acknowledgements: Authors are thankful to Dr. R.C. Agrawal, National Bureau of Plant Genetic Resources, New Delhi, for statistical analysis. Research was supported in part by the Illinois Agricultural Experiment Station and a grant from the Illinois Soybean Program Operating Board. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Buitink J, Glaessens MMAE, Hemminga MA & Hoekstra FA (1998 a) Plant Physiol 118, 531-541. Buitink J, Walters JC, Hoekstra FA & Crane J (1998b) Physiol Plant 103, 145-153. Hymowitz T, Singh RJ & Kollipara KP (1998) in Plant Breeding Reviews, (ed) J Janick, John Wiley and Sons Inc, NY, pp 289-317. Collins FC, Lertmorgkol V & Jones JP (1973) Crop Sci 13, 493-494. Hymowitz T, Wolley JT & Peters DB (1987) Soybean Genet Newsl 14, 271-272. Inagaki M (2000) in Cryopreservation of Tropical Plant Germplasm: current research progress and application, (ed) F Engelmann & H Takagi, International Plant Genetic Resources Institute, Rome, Italy, pp 130-135. Johri BM & Vasil IK (1961) Botanical Review 27, 325-381.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Kenworthy WJ (1989) in World Soybean Research Conference IV: Pcoceedings, (ed) AJ Pascale, Asco Argentina de la Soja, Buenos Aires, pp 883-888. Lim S & Hymowitz T (1987) Plant Dis 71, 891-893. Mignucci JS & Chamberlain DW (1978) Phytopathology 68, 169-173. Ng NQ & Daniel IO (2000) in Cryopreservation of Tropical Plant Germplasm: current research progress and application, (ed) F Engelmann & H Takagi, International Plant Genetic Resources Institute, Rome, Italy, pp 136-139. Pereira TNS, Ilarslan H & Palmer RG (1997) Genome 40, 273-285. Rajashekharan, PE & Ganeshan S (1994) J Horticult Science 69, 565-569. Schoen, DJ, Burdon JJ & Brown AHD (1992) Theor Appl Genet 83, 837-832. Shivanna, KR & Johri BM (1985) The Angiosperm Pollen: structure and function, Wiley Eastern Ltd., New Delhi. Shivanna KR & NS Rangaswamy (1992) Pollen Biology: a laboratory manual, SpringerVerlag, Berlin. Shivanna, KR, Linskens HF & Cresti M (1991) Theor Appl Genet 81, 38-42. Singh BB, Gupta SC & BD Singh (1974) Indian J Genet Plant Breed 34, 400-404. Singh RJ, Kollipara KP & Hymowitz T (1993) Crop Sci 33, 1002-1007. Stanley RG & Linskens HF (1974) Pollen Biology, Biochemistry and Management. Springer-Verlag, Berlin. Towill LE & Walters C (2000) in Cryopreservation of Tropical Plant Germplasm: current research progress and application, (eds) F Engelmann & H Takagi, International Plant Genetic Resources Institute, Rome, Italy, pp 115-129. Towill LE (1985) in Cryopreservation of Plant Cells and Organs, (ed) KK Kartha. CRC Press, Boca Raton, pp 171-198. Walters C (1998) Seed Sci Res 8, 223-244. Withers LA (1991) in Plant Genetic Resources – conservation and management, concepts and approaches, (eds) RS Paroda & RK Arora, IBPGR Regional Office for South and Southeast Asia, New Delhi, pp 273-298. Accepted for publication 4/4/03
