Department of Botany, University of Port Elizabeth, South Africa.

Gibberellin and Stratification Required for the Germination of Erica junonia, an Endangered Species

J. G. C. SMALL and CORNELIA J. GARNER

Received January 23, 1980 . Accepted February 20, 1980

Summary

Erica junonia seeds are dormant. Gibberellic acid promoted germination only when combined with stratification at 5°C. GA7 was more effective than GA3• A preincubation (4 weeks) at 23115 cC prior to chilling enhanced germination considerably. Without GA, chilling had little germination promoting effect. A number of other treatments including various levels of N02-, N03-, ethrel, ethylene, thiourea, H 20 2, oxygen, kinetin, kinetin/GA combinations, scarification and a range of constant and fluctuating temperatures, proved inadequate to promote germination.

Key words: Germination, gibberellin, stratification, Erica junonia.

Introduction

Erica junonia BOLUS, one of the most beautiful ericas having the largest flowers in the genus, is restricted to a single mountain peak in the Cold Bokkeveld, Cape Province, South Africa (BAKER and OLIVER, 1967). Only a few hundred plants were observed by one of us (JGCS) on a recent visit to the natural habitat.

According to WINTER (Chief Curator, National Botanic Gardens, Kirstenbosch -personal communication) attempts at propagating plants from seed have been unsuccessful, due to lack of germination.

Due to its restricted numbers and distribution as well as the difficulty in propaga­tion, E. junonia has been declared an endangered species (ROBERTSON and McNAUGHTON, 1978). For conservation of this species a knowledge of its germina­tion requirements appears important.

A recent investigation has shown no abnormalities in embryo-sac development (ROBERTSON and McNAUGHTON, 1978). Mature seeds contain what appears to be normal embryos (unpublished results).

The present investigation shows that seeds are dormant and require a combination of warm and cold stratification as well as gibberellin to trigger germination.

Materials and Methods Seeds from freshly dehisced ovaries were collected from plants in the natural habitat and

stored at 21°C in screw capped glass bottles after one week of air drying.

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180 ]. G. C. SMALL and CORNELIA]' GARNER

The standard germination procedure consisted of incubating 25 surface sterilized seeds on two layers of Schleicher and Schiill No 595 filter paper in 3 ml of distilled water or test solution, contained in 70 mm pyrex petri dishes.

Seeds were sterilized by consecutive immersion in 70 % ethanol for 1 min and in 0.2 %

HgCl2 for 2 min followed by washing with sterile distilled water. Glassware was autoclaved for 3 min at 120°C. Gibberellin and kinetin solutions were sterilized by autoclaving for 15 min at 100°C. Other test solutions were sterilized by filtration. Gibberellins (GA3' Koch-Light; GAr> Nutritional Biochemicals Corporation) were first dissolved in a minimal quantity of 95 % ethanol and kinetin (Sigma) in 0.05 N NaOH before diluting with distilled water.

Warm stratification occurred in the dark at 23/15 ± 0.5 °C (12 h cycle) and cold stratification (called chilling from now on) at 5.5 ± 0.5 °C in Controlled Environments growth cabinets. After chilling, seeds were transferred to the 23/15 °C temperature regime. Treatments were replicated three times.

Results

Although seeds reacted positively to tetrazolium staining, no seeds (sulphuric acid scarified and unscarified) germinated even after eight months when incubated at various constant (10-30°C) and fluctuating (15/10, 20/10, 25/10, 30/10, 20/15, 25/15 and 30/15°C) temperatures. Similary no germination occurred when seeds were incubated at 23/15 DC, under both light and dark conditions, in various levels of N02-, N03-, ethrel, ethylene, thiourea, oxygen, and H 20 2• After finding that isolated embryos did not grow on agar nutrient media of various composition (WHITE, 1963) at 23/15°,21 ° and 25°C it was concluded that embryo dormancy existed.

Subsequent to the above studies the effect of kinetin, gibberellins and stratification was tested. Kinetin at 0,05 mM caused 9,3 % germination when seeds were incubated at 23/15 DC. No further increases were obtained when kinetin was combined with

Table 1: Effect of gibberellin, warm- and cold (chilling) stratification on germination after a total of 21 weeks incubation.

Warm strati- Chilling GA3mM GA7mM fication period period 0 0.005 0.05 0.5 5.0 0.05 prior to chilling

Weeks Weeks 0/0 Ufo Ofo Ofo Ofo Ofo 0 0 0 0 0 0 0 0 0 4 0 4.3 ± 0.2 6.7±6.6 5.3 ± 2.7 0 8.2±6.1 0 8 0 6.6± 3.4 6.6 ± 3.4 17.4± 5.3 0 11.2±7.7 0 12 0 8.5 ± 2.3 13.7±5.6 28.5± 4.1 10.6 ± 6.7 54.7±7.4

4 0 0 0 0 0 0 0 4 4 0 14.2± 2.3 14.2±3.6 26.2± 2.9 9.4±2.8 26.2 ± 8.7 4 8 0 28.1 ± 1.9 42.8±4.4 59.8± 13.3 6.1 ± 1.9 72.0± 8.4 4 12 11.6± 1.6 38.4± 11.0 44.3 ± 1.9 83.8± 9.9 11.0±1.9 87.0±9.2

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Germination of Erica junonia 181

either GA3 or stratification. Gibberellins, however, were found to be very efficient in breaking dormancy when combined with stratification (Table 1).

Without chilling, GA was ineffective in promoting germination. Similarly chilling was largely ineffective when GA was absent; the only exception to this being 12 weeks chilling when preceded by warm stratification. Very striking was the marked promotive effect of warm stratification prior to chilling.

The optimum GAa concentration was 0.5 mM. GA7 (only tested at 0.05 mM) was far more effective than the same concentration of GA3.

The results shown in Table 1 were obtained with freshly harvested seeds. Similar results were obtained with 15 month old seeds, indicating that the dormancy and germination requirements did not change with dry storage.

Discussion

A link between GA, cold stratification (chilling) and breaking of dormancy in

seeds has been known for some time. Exogenous GA can substitute for a chilling requirement in some seeds (CHEN, 1975). This implies that chilling causes a production of GA. There are many reports which relate low temperature treatment of dormant seeds to increased levels of GA-like substances (see JONES and STODDART, 1977). WILLIAMS et al. (1974) have clearly shown that chilling causes increased GA synthesis in hazel seeds upon transfer from the chilling to the germination temperature.

The situation in Erica junonia appears to be fundamentally different from that described above in that neither GA nor stratification are effective alone.

The function of warm stratification in E. junonia requires further investigation. Usually warm stratification prior to chilling is required by seeds which have some or other form of morphologically underdeveloped embryo e.g. Fraxinus excelsior

(VILLIERS, 1972) Panax ginseng (NIKOLAEVA, 1977). In the case of the latter species, where warm stratification is required for hypocotyl development, GA accelerates processing through this stage but cannot replace the cold requirement (NIKOLAEVA, 1977). Erica junonia shows some similarity with Panax ginseng in that GA does not substitute for chilling but differs because chilling without GA, even after warm stratification, is largely ineffective.

The fact that GA, was more effective than GA3 in this study is not surprising. A number of cases have been reported in which GA's other than GA3 (particularly GA4 and GA7) have been more effective (TOOLE and CATHEY, 1961; BRIAN et al., 1962; DURLEY et al., 1976; KARSSEN, 1976).

Whether the complicated requirement for germination has any bearing on the restricted numbers and distribution of E. junonia remains to be investigated. Under

natural conditions the chilling requirement would normally be met since plants are always covered in snow during winter (BAKER and OLIVER, 1967). The source of or method of obtaining additional requirements, indicated as GA in this report, are unknown.

Z. Pjlanzenphysiol. Eg.99. S. 179-182. 1980.

182 ]. G. C SMALL and CORNELIA]' GARNER

The possible role of naturally occurring fungi as a source of GA IS being investigated.

Acknowledgements

This work was supported by research grants from the Council for Scientific and Industrial Research, Pretoria and the University of Port Elizabeth.

References

BAKER, H. A. and E. G. H. OLIVER: Ericas in Southern Africa. Purnell, Cape Town, 1967. BRIAN, P. W., H. G. HEMMING, and D. LOWE: Nature 193, 946 (1962). CHEN, S. c.: Role of gibberellins in dormancy and seed germination. In: KRISHNAMOORTHY,

H. N. (Ed.): Gibberellins and Plant Growth, 91-99. Wiley Eastern, New Delhi, 1975. DURLEY, R. C, J. D. BEWLEY, r. D. RAIL TON, and R. P. PHARIS: Plant Physiology 57, 699

(1976). JONES, R. L. and ]. L. STODDART: Gibberellins and seed germination. In: KHAN, A. A.

(Ed.): The Physiology and Biochemistry of Seed Dormancy and Germination, 77-109. North-Holland, Amsterdam, 1977.

KARSSEN, C. M.: Physiol. Plant. 36, 264 (1976). NIKOLAEVA, M. G.: Factors controlling the seed dormancy pattern. In: KHAN, A. A. (Ed.):

The physiology and Biochemistry of Seed Dormancy and Germination, 51-74. North­Holland, Amsterdam, 1977.

ROBERTSON, B. L. and]. E. McNAUGHTON: JI. S. Afr. Bot. 44, 97 (1978). TOOLE, V. K. and H. M. CATHEY: Proc. Am. Soc. Hortic. Sci. 36, 663 (1961). VILLIERS, T. A.: Seed dormancy. In: KOZLOWSKI, T. T. (Ed.): Seed Biology 2, 219-281.

Academic Press, New York, 1972. WHITE, P. R.: The Cultivation of Animal and Plant Cells. Ronald Press, New York, 1963. WILLIAMS, P. M., ]. W. BRADBEER, P. GASKIN, and J. MACMILLAN: Planta (Bed.) 117, 101

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Prof. J. G. C SMALL, Department of Botany, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa.

z. Pflanzenphysiol. Bg.99. S.179-182. 1980.