Cytological Analysis of Embryogenic Callus and Regenerated Plants of Urginea Indica Kunth., Indian Squill

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Caryologia International Journal of Cytology, Cytosystematics and

Cytological Analysis of Embryogenic Callus and Regenerated , Indian Squill,

10796964 To link to this article: https://doi.org/10.1080/00087114.1989.

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Cytological Analysis of Embryogenic Callus and Regenerated Plants of Urginea Indica Kunth., Indian Squill Sumita Jha To cite this article: Sumita Jha (1989) Cytological Analysis of Embryogenic Callus and Regenerated Plants of Urginea Indica Kunth., Indian Squill, Caryologia, 42:2, , DOI: / To link to this article: Published online: 30 Jan Submit your article to this journal Article views: 163 View related articles Citing articles: 5 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 20 December 2017, At: 12:27

CARYOLOGIA Vol. 42, n. 2: 165-173, 1989 CYTOLOGICAL ANALYSIS OF EMBRYOGENIC CALLUS AND REGENERATED PLANTS OF URGINEA INDICA KUNTH.

2 CARYOLOGIA Vol. 42, n. 2: , 1989 CYTOLOGICAL ANALYSIS OF EMBRYOGENIC CALLUS AND REGENERATED PLANTS OF URGINEA INDICA KUNTH., INDIAN SQUILL SUMITA JHA Centre of Advanced Study, Department of Botany, University of Calcutta, Calcutta , India. INTRODUCTION SUMMARY - Friable calli were induced from bulb scale explants of diploid (2n = 20) Indian squill, Vrginea indica Kunth. Embryogenic calli were formed when one year old friable calli were allowed to remain on the high 2,4-D medium for a prolonged period. The original explant, friable calli, somatic embryos and plant regenerated via somatic embryogenesis were examined cytologically. The original explant had a negligible level of DNA variation. The dedifferentiating calli showed complete elimination of diploid cells after 6 months and friable calli were composed of cells with high chromosome number ( ). Embryogenic calli contained large and small cells, the latter with deeply stained nuclei, showing variable ploidy (6n-12n). Globular embryoids were also composed of polyploid cells, with chromosome numbers ranging from with a few highly polyploid cells(> 160). Among about a thousand plants regenerated from these friable embryogenic cultures, chromosome numbers of 150 plants were determined. All plants were polyploid. About 50% of the plants were octaploid; 6% were predominantly hexaploid and rest were of variable ploidy. The results show that highly polyploid cells are capable of forming somatic embryoids and regeneration. Urginea indica Kunth., commonly known as Indian squill, is medicinally important for its bufadienolide content GHA and SEN 1981, 1983a). Plant regeneration through organogenesis in this species GHA et al. 1984) revealed the majority of plants to be diploid with a few tetraploids GHA and SEN 1987a, 1987b). Plant regeneration through somatic embryogenesis from long term culture has recently been reported in this species GHA and SEN 1986). Somatic embryogenesis has been reported in only three other species of Liliaceae viz., Allium sativum (ABo EL-NIL 1977), Asparagus officina/is (REuTHER 1977a, 1977b) and Hemerocallis sp. (KruKoRIAN and KANN 1981a). Cytogenetic analysis of plants regenerated through somatic embryogenesis in Hemerocallis revealed all the plants to be karyotypically stable (KRIKORIAN and KANN 1981b).

166 JHA In garlic, plants regenerated from callus cultures included tetraploids, aneuploids and mixoploids in addition to normal diploids (DoLEZEL et al. 1986).

3 166 JHA In garlic, plants regenerated from callus cultures included tetraploids, aneuploids and mixoploids in addition to normal diploids (DoLEZEL et al. 1986). This paper describes the results of cytological study of embryogenic cell cultures and plants of U. indica regenerated by somatic embryogenesis. MATERIALS AND METHODS Tissue culture. - A high yielding diploid (2n = 20) cytotype (JHA and SEN 1983b) of Urginea indica Kunth. collected from Tuticorin was used in this study. Callus was established from bulb explants on modified MS medium supplemented with 2 mgl- 1 2,4-D + 15% coconut milk (CM, v/v). The detail of the procedure has been reported in earlier papers (JHA et al. 1984; }HA and SEN 1986). The soft friable calli obtained were maintained for a year in three concentrations of 2,4-D - 0.5, 1 and 2 mgl- 1 and regularly subcultured every four weeks.' Embryogenesis was not noted during this period. After a year, calli from these three sets were subcultured in 500 ml flasks containing 200 ml solidified MS medium supplemented with 0.5, 1 and 2 mgl- 1 2,4-D and transferred at 6 month intervals. Formation of embryogenic clumps were noted in 40% of calli growing in the presence of 2 mgl- 1 2,4-D between the 2nd and 4th year after callus induction. The globular embryoids were induced to develop into complete bulbous plants with subsequent transfer to MS medium containing mgl- 1 BAP for 4-6 weeks and then to MS medium mgl- 1 NAA mgl- 1 Kinetin for 8 weeks (JHA and SEN 1986). A final period in liquid MS medium stimulated shoot and root growth simultaneously. Cytological analysis. - Callus tissues were prepared for cytological analysis by taking samples from a wide spatial range of each culture to ensure independence and to estimate the total range of variability as accurately as possible. Chromosome counts were taken from cells of each culture age. Callus (embryogenic and nonembryogenic) and globular embryoids were fixed in chilled Carnoy's fixative for overnight and stained with orcein-hcl (9:1) mixture and squashed with 45% acetic acid. Root-tips from regenerated plants were pretreated with 0.05% colchicine for 2 hours prior to fixation. For DNA cytophotometry, the explants were hydrolysed in 5N HCl for 20 min at room temperature and nuclei stained with Feulgen reagent. DNA contents of nuclei (at least 50 nuclei for each observation) were measured in a Reichert Zetopan with microphotometer and transformed to C values by taking as standard root apex prophases ( = 4C) of control plants as has been followed in other studies (D' AMATo 1975). RESULTS Nuclear condition of original explant. - The nuclear DNA content of the cells comprising the bulb scale tissue was found to be distributed in one peak

4 REGENERATED PLANTS OF URGINEA INDICA 167 corresponding to the 2C (G1) DNA content. After 6-8 days of culture, a second peak of cells with doubled DNA content (4C) appeared indicating the process of nuclear DNA synthesis. Cells with intermediate DNA values were observed in low frequency during later period. The cells with 8C level occurred at very low frequency. Nuclear condition of non-embryogenic calli. - White friable calli were composed of large parenchymatous cells only, a high frequency of binucleate and multinucleate cells were noted. Mitotic index was low. While one month old calli contained diploid cells (98%) (Fig. 1) with a few polyploid cells, diploid cells were completely eliminated after 7-8 months of culture and calli were predominantly composed of cells of chromosome numbers ranging from (Fig. 2) after 1 year (Table 1). TABLE 1 - Chromosome number and frequency of cytological anomalies at anaphase/telophase in nonembryogenic calli, embryogenic calli and globular embryoids. Calli growing in presence of 2,4- D 2 mgr 1. (Chromosome counts from cells of each culture age). Chromosome % of anaphase/telophase cells exhibiting Age of numbers Nature of calli callus observed micro- budlags multi- multi- (% to total) nuclei ding polar nucleate 1 month Non-embryogenic 20 (98%) 2% 40 (2%) 1 years Non-embryogenic (80%) 1% 1% 4% 8% (20%) 2 years Non-embryogenic 300 (66%) 3% 6% 2% 5% (34%) 2 years Embryogenic 6 months (a) Large cells 300 (32%) 3% (28%) (40%) 20% (b) Small 120 (5%) 7% 4% 6% 12% meristematic (20%) cells (45%) 80 (16%) (14%) 2 years Globular 120 (2%) 12% 5% 1% 2% 15% 8 months embryoids (5%) (43%) 80 (20%) (19%) 60 (11%)

168 JHA Fig. 1. - A diploid metaphase plate from one month old calli. Fig. 2.

5 168 JHA Fig A diploid metaphase plate from one month old calli. Fig Highly polyploid metaphase plate from 2 year old non-embryogenic calli showing -200 chromosomes. Figs Nuclear abnormalities in embryogenic calli multipolarity (3, 4), tetranucleate (5) and micronuclei formation (6).

REGENERATED PLANTS OF URGINEA INDICA 169 Embryogenic calli. - Somatic embryogenesis in U. indica occurred in a discrete sector of the callus and not from entire callus tissues.

6 REGENERATED PLANTS OF URGINEA INDICA 169 Embryogenic calli. - Somatic embryogenesis in U. indica occurred in a discrete sector of the callus and not from entire callus tissues. The initial morphological signs of embryogenesis were the greening of surface zones of calli between the 4th and 5th month of the 3rd six monthly subculture (callus age 2 years 4 months). Such green zones (pre-embryogenic) were composed of large and small cells. The large vacuolated parenchymatous cells were nonembryogenic containing high frequency of multinucleate cells (Table 1) and very few cells were dividing (MI 0.1%). The small cells were round, richly cytoplasmic with prominent nuclei. They were meristematic showing high MI (5%) and various types of nuclear abnormalities (Figs 3-6). Chromosome number counts revealed large cells had chromosomes approximately while in small meristematic cells the number ranged from 60 to 120. Globular embryos. - These were composed of cells with a chromosome number ranging from 60 to 90 and also a few highly polyploid cells. Other mitotic abnormalities included micronuclei (1-3) in 5-10% cells. Nuclear budding was also a characteristic feature of these cells. Regenerated plants. - Plants regenerated from somatic embryos were grouped into two types; those showing normal leafy shoots with bulbs and those without bulbs and with curled stunted leaves. An analysis of chromosome number did not show any difference in these two groups of plants. Plants that gave rise to bulbs were separated and chromosome number counts from root tips of 150 such plants were determined. All plants were polyploid, 50% were predominantly octaploid with a low frequency of cells with other higher chromosome numbers; 6% of the plants were predominantly hexaploid whereas the rest of the plants showed variable chromosome numbers (Table 2, Figs. 7-10). TABLE 2 Chromosome number counts in root tips of plants regenerated from somatic embryos derived from 2 year old bulb scale calli. Regenerated plants Most frequent (60-75%) Other chromosome metaphases-chromosome numbers (5-30% of No. (%) number metaphases analyzed) 9 (6) 60 >50< 60 >60< (50) 80 >60< 80 >100< (44) >100<120 >80<100

170 JHA Figs. 7-10. -Metaphase plates from root tips of regenerated plants showing 60, 64, 70 and nearly 80 chromosomes respectively. DISCUSSION Somatic embryogenesis in U.

7 170 JHA Figs Metaphase plates from root tips of regenerated plants showing 60, 64, 70 and nearly 80 chromosomes respectively. DISCUSSION Somatic embryogenesis in U. indica occurred in discrete sectors (green zones) of the callus and not from all callus tissues, thus the calli exhibited variability in their ability to regenerate plants. Embryogenic calli and well developed embryoids were noted when, 2,4-D in the medium was gradually lowered by prolonged growth of the callus on the same medium without subculture. Changes in the ploidy level have been noted in many cultures leading to mixed populations of polyploids and aneuploids and plant regenerated from such cultures often show a range of chromosome complements (D'AMATO 1978). The appearance and extent of variability in plant cells grown in culture may depend on the explant used; the culture conditions employed (D' AMATO 1978) and the method of plant regeneration (VAsiL 1983). The bulb explants used as source material for aseptic cultures in the present study had a negligible

REGENERATED PLANTS OF URGINEA INDICA 171 level of DNA variation. There was a rapid elimination of diploid cells and stimulation of polyploid mitosis in the dedifferentiating cultures of U.

8 REGENERATED PLANTS OF URGINEA INDICA 171 level of DNA variation. There was a rapid elimination of diploid cells and stimulation of polyploid mitosis in the dedifferentiating cultures of U. indica either by chromosome endoreduplication in vitro or by formation of a restitution nucleus. The change from diploid to polyploid took place very rapidly and suggests that these polyploid cells had a positive selective value with respect to normal cells in the conditions in vitro. The polyploid cells had a division mechanism as efficient as normal diploid cells as anaphase abnormalities were rare in the dedifferentiated calli. These highly polyploid (chromosome no ) calli did not show any sign of morphogenesis for two years. The two year old calli, maintained on a medium with 2,4-D and subcultured at six month intervals showed development of embryogenic calli. The embryogenic calli were also composed of highly polyploid cells. However, the variation in chromosome number in the small meristematic cells ranged from whereas the large non-embryogenic cells showed chromosomes. In addition various types of nuclear abnormalities were noted in high frequency with onset of embryogenesis. These observations suggest that the shift from dedifferentiation to differentiation in such long term calli was accompanied by somatic reduction in chromosome number showing the tendency of embryogenic calli to stabilize towards a lower chromosome number. The lowest chromosome number observed in embryogenic calli is 60 (6n) and the lowest in non-embryogenic calli is 120 (12n). Thus somatic reduction by elimination of chromatin through various ways must have played a determining role in bringing about this shift in morphogenesis. The apparent reduction in polyploidy could be as a result of a mechanism leading to chromosome reduction or alternatively as a result of cells with lower ploidy levels becoming predominant as a result of different growth rates. The globular embryoids also showed this variation although very high chromosome numbers ( > 120) were not found to occur. Nuclear processes leading to lowering of chromosome number were evident during development of embryoids also. The regenerated plants, as expected, are polyploid. Thus the generally held view that the embryogenic cell cultures are largely stable cytologically (SWEDLUND and VASIL 1985) is not applicable to this species. The calli, in this species, were composed of cytologically atypical cells (VAsiL 1983), yet they are capable of forming somatic embryos and phenotypically normal plants. The embryogenic capacity of cultures has been seen to decrease and disappear during progressive subculturing (SYONO 1965) and this loss of potential has been traced, at least in certain cases, to the change in chromosome complement where aneuploids gradually replace diploids (SMITH and STREET 1974). By changing the sequence of growth regulators with each subculture, a non-embryogenic line of Daucus carota gradually regained embryogenic capacity (CHANDRA 1981). Whether this was due to selective enrichment of preexisting embryogenic cells in suspension or the reinduction of cells that are epigenetically changed was not determined. In U. indica, however, the non-

172 JHA embryogenic cells were induced to develop somatic embryos and although all the cells comprising embryogenic calli were highly abnormal as far as chromosome number is concerned, they gain the

9 172 JHA embryogenic cells were induced to develop somatic embryos and although all the cells comprising embryogenic calli were highly abnormal as far as chromosome number is concerned, they gain the capacity of embryogenesis thereby showing that the change was not epigenetic. In studies of somatic embryogenesis in long term Daucus cultures, embryos and plants could be grown but they were sterile (SussEX and FREI 1968). Acknowledgement. -I wish to express my best gratitude to Prof. Sumitra SEN, CAS, Dept. of Botany, Calcutta University for valuable advice and discussion and to Prof. A.K. SHARMA, Programme Co-ordinator, for facilities provided. The financial assistance of University Grants Commission is gratefully acknowledged. REFERENCES ABo EL-Nn.. N.M., Organogenesis and embryogenesis in callus cultures of gprlic (Allium sativum L.). Plant Sci. Lett., 9: CHANDRA N., Clonal variation in morphogenetic response of cultured cells of carrot, Daucus carota L. Ind. J. Exp. Biol., 19: D'AMATO F., In crop genetic resources for today and tomorrow. 0. Frankel and J.G. Kawkes eds., pp , Cambridge Univ. Press, London. -, Chromosome number variation in cultured cells and regenerated plants. In «Frontiers of plant tissue culture». T.A. Thorpe ed., pp , Calgary Press, Canada. DoLEZEL J., NovAK F.J. and HAVEL L., Cytogenetics of gprlic (Allium sativum L.) in vitro culture. In «Nuclear techniques in vitro culture for plant improvement», pp , International Atomic Energy Agency, Vienna. ]HA S. and SEN S., Bufadienolides in different chromosomal races of Indian squill. Phytochemistry, 20: , 1983a. -A quantitation of principal bufadienolides in different cytotypes of Urginea indica. Planta Medica, 47: , 1983b. - Chromosome study of diploid Indian squill. Cytologia, 48: , Development of Indian squill (Urginea indica Kunth.) through somatic embryogenesis from long term culture. J. Plant. Physiol., 124: , 1987a.- Nuclear changes and organogenesis during callus culture of Urginea indica Kunth. Cytologia, 52: , 1987b. - Karyotype variability in regenerated plants of Urginea indica Kunth. Cytologia, 52: ]HA S., MITRA G.C. and SEN S., In vitro regeneration from bulb explants of Indian squill Urginea indica Kunth. Plant Cell Tissue and Organ Culture, 3: KluKoRION A.D. and KANN R.P., 1981a. - Plantlet production from morphogenetically competent cell suspension of day lity. Ann. Bot., 47: KluKoRION A.D., STAICU S.A. and KANN R.P., 1981b. -Karyotype analysis of a Day lity clone reared from aseptically cultured tissues. Ann. Bot., 47: MURASIDGE T. and SKOOG F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: REUTHER G., 1977a. - Adventitious organ formation and somatic embryogenesis in callus cultures of Asparagus and Iris and its possible application. Acta Hortic., 78: , 1977b. - Embryoide Differenzierungsmuster im Kalus der Gattungen Iris und Asparagus. Ber. Dtsch. Bot. Ges., 90: SMITH S.M. and STREET H.E., The decline of embryogenic potential as callus and suspension cultures of carrot (Daucus carota L.) are serially subcultured. Ann. Bot., 38:

10 REGENERATED PLANTS OF URGINEA INDICA 173 SussEX I.M. and FREI K.A., Embryoid development in long term tissue cultures of carrot. Phytomorphology, 18: SWEDLUNG B. and VAsiL I.K., Cytogenetic characterization of embryogenic callus and regenerated plants. Theor. Appl. Genet., 69: SYONO K., Changes in organ forming capacity of carrot root callus during subcultures. Plant Cell Physiol., 6: VAsiL I.K., Regeneration of plants from single cells of cereals and grasses. In «Genetic Engineering in Eukaryotes», Edt. P.F. Lurquin and A. Kleinhops, pp , Plenum Publishing Corporation. Received 16 May 1988; accepted 10 October 1988

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