Dormancy break in seeds of Impatiens glandulifera Royle

Dormancy break in seeds of Impatiens glandulifera Royle BY PAULINE M. MUMFORD* School of Biological Sciences, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. (Received 27 April 1989; accepted 14 December 1989) SUMMARY When dormancy is broken by chilling in Impatiens glandulifera Royle anthocyanin appears in the cells of the root cap acting as a marker of the physiological condition of the seeds. This precedes growth of the embryo as indicated by increase in seed weight. Abscisic acid at some concentrations can inhibit dormancy break and anthocyanin synthesis in the root cap. All tissues of the embryo, when excised, responded independently to the cold stimulus. Grafting pieces of dormant and non-dormant seed showed the agent effective in breaking dormancy was not transmissible between a piece of chilled embryo placed in contact with a piece of dormant embryo. Key words: Embryo dormancy, ABA, anthocyanin synthesis. and progressing in the cotyledons. However proteolysis and starch decrease also occur in imbibed Many seeds require a period of chilling after seeds at higher temperatures so these changes may imbibition to break dormancy and aspects of the be a direct consequence of imbibition rather than changes induced by cold temperature have been low temperature treatment (Bouvier-Durand et al., studied in various species but the mechanism is still 1984). Although these studies indicate where some of the poorly understood. Early work suggested that removal of the testa or pericarp could remove or early changes in dormant seed may occur they reduce the chilling requirement (Bradbeer, 1968) provide no evidence of where in the seed the cold implicating chemical inhibitors in the seed coverings. stimulus is perceived or whether the transmission of Removal of the testa is effective in stimulating some a message is involved from one region of the seed to growth in many rosaceous species but seedling the embryonic tissues. Recently attention has been development is not normal indicating some in- centred on changes within the nuclei of cells hibitory mechanism still remains in the embryo following chilling. In Acer platanoides rrna accu(stokes, 1965). The role of abscisic acid (ABA) in mulates in the cells of the embryonic axis and RNA seed dormancy has been reviewed by Walton (1981) polymerase activity gradually increases with chilling and while in many cases a decrease in ABA is time (Slater and Bryant, 1987) suggesting more associated with the loss of dormancy there are ribosomes may be available for the translation of inconsistencies in the relationship between ABA mrna. In most studies of dormancy the criterion used to levels and the consequences of chilling. A histological approach was made to the problem assess dormancy break is the rupture of the testa of what changes occur in apple seeds on chilling by resulting from the initiation of growth of the radicle Dawidowicz-Grzegorzewska and Zarska-Macie- or hypocotyl of the embryonic axis. However jewska (1979) and Dawidowicz-Grzegorzewska Impatiens glandulifera seeds, which require a chilling (1981) who suggested that chilling results in the period (Mumford, 1988) afford an exceptional opbreakdown of carbohydrate and protein reserves portunity to detect dormancy break following chillstarting in the central region of the embryonic axis ing, before growth is involved, and therefore this species has been used to investigate some of the early * Present address: 60, Odell Place, Edgbaston, Birmingham events of loss of dormancy. B5 7RQ. INTRODUCTION

172 Pauline M. Mumford {d) transversely removing the cotyledon apex; {e) transversely removing the root tip. Impatiens glandulifera Royle seed which had been Control seeds were left intact. Seeds were placed in stored since 1985 at low moisture content at 4 C and petri dishes containing moist filter paper and placed whose chilling requirement was known to be within at 4 C for 14 days. The ability of parts of non-dormant seeds to the range of 7 to 14 days (Mumford, 1988) was used. The testa was removed from some seeds to allow influence the tissues of dormant seeds and vice versa observation to be made of visible changes in the was tested by taking stratified seeds whose testas appearance of the seeds as they emerged from were beginning to rupture, sectioning them and then matching the cut surface of the non-dormant seed to dormancy. The pattern of water uptake of seeds as dormancy its mirror image of a dormant seed. Seeds were was broken was monitored by placing seeds in sectioned longitudinally through and between the compartmentalised petri dishes, one seed per com- cotyledons, transversely through the cotyledons, the partment, on moist filter paper and individual seeds root tip or cotyledon apex was excised, or half of one were blotted dry and weighed on a daily basis for at cotyledon was removed. The 'hybrid seed' was then least 20 days. The filter paper was moistened with either embedded in agar or placed on moist filter either water or abscisic acid at a range of concen- paper at 20 C and development observed. trations from lo"** M to 10"^ M. Seeds were kept at 4 C and a water control treatment was maintained at RESULTS 20 C. Twenty seeds per treatment were weighed. To examine the role of different parts of the seed The seed of Impatiens glandulifera consists of a in the perception of the cold stimulus seeds were brown patterned testa and an embryo comprising allowed to imbibe for one hour, so that the tissues two fieshy cotyledons and a small embryonic axis. were soft, and then they were sectioned in the The plumular meristem, just visible at a magnification of X 40, is embedded at the base of the following ways: {a) longitudinally through the centre of both cotyledons and the radicle is short and distinct. The embryo is uniformly white when first imbibed. As cotyledons; stratification proceeds the radicle region becomes (6) longitudinally between the cotyledons; slightly yellow and when dormancy is broken (c) transversely through the centre of the seed; MATERIALS AND METHODS 8 12 16 20 0 4 8 12 16 20 Imbibition time (days) 0 8 16 20 Figure 1. Changes in the weights of individual imbibed over a 20 dayy pperiod at: (A) ividual seeds im () 4 C, water; (B) 4 C, ABA 10-**M; ( C ) 4 C, ABA IO-^ 10^M; ( E ) 4 C, C ABA 10'^ M; ( F ) 20 C, C water. water V (D D ) 4 C, ABA 10"^ indicates the appearance of anthocyanin in the root cap.

Dormancy break in seeds of Impatiens glandulifera 173 Table 1. The response of different parts of the seed to chilling at 4 C Response Part of seed* (c) id) («) Control (e) 0 Anthocyanin in root cap Production of secondary roots Hypocotyl elongation Chlorophyll in cotyledons Cotyledon enlargement - Poor Poor -I- 1 -I- - - - * See 'Materials and Methods'. anthocyanin gradually appears in the tip of the radicle region. All root tips of the pigmented plants of /. glandulifera contain anthocyanin but the pigment is restricted to the cells of the root cap. This distinctive feature in the development of the seeds provides a convenient marker of their physiological status. If seeds are transferred to a higher temperature (20 C) at the time of the appearance of pigment in the radicle they will germinate but transfer before this stage generally results in increased dormancy. Within a few days of the development of anthocyanin in the root cap, the radicle region enlarges slightly and the hypocotyl starts to elongate. At this point the testas of intact seeds rupture. This is followed by the emergence of four pigmented areas, arranged in a collar around the radicle w^hich develop into secondary roots. High temperature considerably accelerates the elongation of the hypocotyl and roots and subsequently the cotyledons change from white to yellow and on exposure to light produce chlorophyll. By contrast dormant embryos remain white and constant in size. Removing the testa from seeds did not affect their response to temperature indicating that embryonic dormancy is being broken by low temperture. The pattern of change in seed weight over a period of 20 days imbibition is shown for five typical seeds subjected to different treatments (Fig. 1) and indicates that moisture is taken up at a similar rate during the first 24 hours by all seeds irrespective of their chemical medium, and seed weight generally remains constant until after dormancy has been broken. Chilling broke dormancy of seeds in water and at low concentrations of ABA, lo"** and 10'^ M. An increase in seed weight, attributable to water uptake and growth, usually occurred 2-3 days after anthocyanin appeared in the root cap. However low concentrations of ABA delayed the time to dormancy break and the delay increased with increasing Figure 2. Parts of dormant and stratified seeds of Impatiens glandulifera grafted together and placed for 4 days at 20 C. Dormant seed appears wbite. Seeds sectioned before grafting as follows: (a) longitudinally through both cotyledons, (b) longitudinally between cotyledons, (c) root tip excised, (d) transversely through cotyledons, (e) transversely removing cotyledon apex and (f) half of one cotvledon excised. concentration. Seeds at 20 C and those in 10 ^ and 10'* M ABA at 4 C remained dormant and showed no significant change in seed weight after the initial phase of imbibition. The production of anthocyanin in the root cap clearly preceded any growth of the embryonic axis. The response of different parts of sectioned seeds

174 Pauline M. Mumford to chilling is shown in Table 1. All parts of the seed responded to the cold period. Seeds without the root region were able to germinate and regenerate roots from the cut end of the elongating hypocotyl and cotyledons developed chlorophyll in the normal way. Pieces of cotyledon detached from the shoot and root meristems, also developed plastids and showed enlargement on transfer to higher temperature although they were not capable of root production. Seeds sectioned directly through the embryonic axis generally produced some roots and expanding green cotyledons from both sides of the seed although none of the seedlings were normal and hypocotyl elongation was severely affected. This indicates that cells from all regions of the seed independently perceive and respond to the cold stimulus. The effects of combining dormant and nondormant seed tissues together are shown in Figure 2. Generally there was no infiuence of one tissue type upon the other or evidence of transmission of metabolites between cells. The excised radicle region from non-dormant seeds when matched with dormant tissues showed limited growth. Cotyledons or parts of cotyledons plus the embryonic axis from dormant seeds showed no signs of development when in contact with non-dormant seed tissues whereas cotyledons and axes from stratified seeds all showed growth or colour changes associated with development regardless of their proportion to the dormant tissues with which they were combined. These results confirm that all parts of the seed respond according to the chilling they have received and that they are not influenced by adjacent cells that are in a different physiological state. DISCUSSION The appearance of anthocyanin in the radicle region of the embryonic axis of /. glandulifera is a convenient indicator that seeds have responded to chilling and are capable of germination on transfer to higher temperatures. The production of anthocyanin is not an essential feature of the dormancy breaking process but rather a response of a certain group of cells to changes induced by the cold period. Anthocyanin is produced in a limited number of cells of the root cap, those normally containing amyloplasts and associated with the perception of gravity. The biosynthesis of anthocyanin requires a source of phosphoenolpyruvate, erthyrose-4-phosphate and acetyl CoA. These metabolites could be derived from starch deposits in the root cap cells. Dawidowicz-Grzegorzewska & Zarska-Maciejewska (1979) suggested that in apple seeds one of the first detectable changes in the embryonic axis at low temperature was a starch decrease. Dormancy break by chilling can be inhibited by ABA if the concentration is sufificiently high, and this is shown by the absence of anthocyanin in the root cap. Studies by Karssen (1976fl, 6) on Chenopodium album seeds, whose dormancy can be broken by light, suggest that ABA inhibits radicle growth but not the rupture of the outer testa or slight enlargement of the radicle. However work on seeds like mustard and rape, which have no natural dormancy mechanism, indicates that ABA can impose dormancy by regulating the water uptake associated with radicle growth (Schopfer, Bajracharya & Plachy, 1979; Schopfer & Plachy, 1984). Results obtained with /. glandulifera confirm that radicle growth and water uptake are inhibited by high concentrations of ABA but events which precede these processes, signalled by the production of pigment in the root cap, are also prevented by ABA suggesting lack of water uptake and growth are only consequences of an earlier metabolic block to the process of germination. It is not known if seeds of /. glandulifera contain ABA which may contribute to the natural dormancy mechanism but it seems unlikely that this is the basis of the dormancy since prolonged chilling can alleviate natural dormancy but cannot stimulate seeds placed in high concentrations of exogenous ABA to germinate. When attempts were made to graft dormant and non-dormant seed tissues there was a lack of transmission of any factors associated with dormancy. The fact that even small pieces of cotyledon showed dormant or non-dormant responses appropriate to their previous stratification treatment suggests that all cells of the seed respond independently to the cold stimulus and therefore a cellular model is required to account for the effects of chilling. The responses to chilling vary with the region of the embryo stratified indicating chilling affects a process common to all cells regardless of their genetic programming and the specific pathways involved w-hich produce the observed effects. One possibility is that low temperture has a direct effect on gene regulation or post-transcription or posttranslation of the genes in the cell. REFERENCES (1968). Studies in seed dormancy. IV. The role of endogenous inhibitors and gibberellin in the dormancy and germination of Corylus avellana L. seeds. Pianta 78, 266-276. BouviER-DuRAND, M., DAWIDOWICZ-GRZEGORZEWSKA, A., THEVENOT, C. & COME, D. (1984). Dormancy of apple embryos. 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