PlantCell Reports

Plant Cell Reports (1996) 15:470-473

9 Springer-Verlag 1996

Production of dodecaploid plants of Japanese persimmon (Diospyros kaki L.) by colchicine treatment of protoplasts Mihoko Tamura 1, Ryutaro Tao 2, and Akira Sugiura 2 1 Department of Biotechnological Science, Faculty of Biology Oriented Science and Technology, Kinki University, Uchita, Wakayama 649-64, Japan 2 Laboratory of Pomology, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-01, Japan Received 3 June 1995/Revised version received 31 August 1995 - Communicated by C. Quiros

cytometry, Ploidy level, Woody plant

dodecaploid individuals. However, no dodecaploid plants have been reported so far within Diospyros. Colchicine treatment of actively dividing cells can induce chromosome doubling and has been used to make plants with doubled chromosome number. Although there are several target tissues for colchicine treatment, such as meristems and seeds, occurrence of cytochimeric plants is often a big problem (Goldy and Lyrene 1984; Awoleye et al. 1994). Colchicine treatment to a protoplast at the very beginning of its division could be one method to overcome the problem because plants can be regenerated from a single cell with doubled chromosome number. Difficulty in plant regeneration from protoplasts, however, is a barrier to utilize the technique and no such attempt has been made so far. Fortunately, an efficient plant regeneration system has been developed for Japanese persimmon protoplasts (Tao et al, 1991a; Tamura et al, 1993). In this paper, we describe regeneration of non-chimeric dodeeaploid persimmon plants from protoplasts treated with colchicine.

Introduction

Materials and methods

Summary. Dodecaploid plants of Japanese persimmon (Diospyros kaki L.) were obtained by colchicine treatment of protoplasts. Callus protoplasts of 'Jiro' (2n=90, x=15) were cultured in modified KM8p medium with 0.1% colchicine for 3-9 days. After colchicine treatment, they were cultured using agarose bead culture. Microcalli were recovered from the protoplasts after 3 months. Flow cytometric measurement showed that nine of 31 callus lines obtained from 6 days of colchicine treatment had twice the nuclear DNA content as non-treated controls. Plantlets were regenerated from the calli with twice the nuclear DNA content. Microscopic observation of root tip cells showed that their somatic chromosome number was 2n--180 (x=15). Compared with 'Jiro', dodecaploid plants had longer stomatal guard cells and lower stomatal densities, consistent with increased ploidy.

Key words: Chromosome doubling, Colchiploid, Flow

Japanese persimmon (Diospyros kaki L.) has long been believed to be hexaploid (2n=90, x=15)(Namikawa, 1930; Namikawa and Higashi, 1928). However, Zhuang et al. (1990a, 1990b, 1992) h a v e r e c e n t l y r e e x a m i n e d chromosome numbers of many Japanese persimmon cultivars and found that some seedless cultivars have nonaploid chromosome numbers of 2n= 135. Since all the nonaploid cultivars bear astringent type fruits, the astringency must be removed by carbon dioxide or alcohol treatment before consumption (Kitagawa and Glucina 1984), which leads to short shelf fife. ff nonaploid cultivars of non-astringent type could be bred, the problem related to the deastringent treatment could be overcome and seedless eultivars with longer sheff life might be produced. One possible method to breed nonaploid or seedless cultivars is to make crosses between hexaploid and Correspondence to." M. T a m u r a

Colchicine treatment of protoplasts and plant regeneration. Callus protoplasts of 'Jiro' were isolated as previously described (Tao et al. 1991a, b). They were embedded in modified KMSp (Kao and Miehayluk 1975; Tamura et al. 1993) agarose medium in a 35-ram petri dish at 5 x 105 cells/ml. The agarose plate was cut into six blocks and transferred to a 60-ram petri dish containing 5 ml of modified KM8p liquid medium with or without 0.1% (w/v) colchicine. The culture was kept at 27~ in the dark on a shaker (50 rpm) for 3 to 9 days. After colchicine treatment, the agarose blocks were transferred to a 90-mm petri dish and washed with 15 ml of fresh modified KM8p medium without colchicine for 1 day on a shaker. After washing colchicine off, the culture was maintained according to the method described earlier to regenerate plants from protoplasts through callus phase (Tao et al. 1991a; Tamura et al. 1993). Three petri dishes were used for each treatment and the experiment was repeated twice in all.

Flow cytometrie analysis. Protoplast-derived calli subcultured for 10 days were cut into pieces in 10raM Tris-HC1 (pH 7.0) containing 0.1% (v/v) Triton X-100, 100mM NaC1, and 10raM Na2EDTA, and kept on

471 ice for 5 rain to release nuclei from cells. The solution was filtered through a 20 y m nylon mesh to remove debris. Nuclei in the filtrate were stained with 100 mg/ml propidium iodide (PI). Relative nuclear DNA content was measured by flow cytometry (Coulter Epics Elite, Florida, USA). Data were analyzed by the Multicycle cell analysis (Phoenix Flow Systems, USA). Chromosome counting. Somatic chromosome number of root tip cells was counted to confirm the ploidy level of plants regenerated from calli with twice the nuclear DNA content as 'Jiro' callus. Root tips were immersed in distilled water at 4~ for 24h and fixed in acetic acid: methanol (1:1) for lb. Then they were hydrolyzed in 5N HC1 at 20~ for 40 rain. After hydrolyzatiou, the root tips were stained in the Feulgen solution at 4~ for 30 min, and washed with 15% (w/v) NaHSO 3 and distilled water. The stained parts of the root tips were excised and put on the slides, covered with an enzyme solution of 4% (w/v) Cellulase RS, 1% (w/v) Pectolyase Y23, 0.07M KC1 and 7.5mM Na2EDTA (pH 4.0), and incubated for 70 min at 37~ After cell wall digestion, they were washed with distilled water, and squashed in acetic acid: methanol (1: 3). Chromosome number was counted using at least 20 cells per line under a microscope. Stoma observation. The abaxial leaf surfaces of the plants regenerated fromprotoplasts were observed with scanning electron microscopy (SEM: $2250N, Hitachi Ltd, Tokyo, Japan). The natural SEM mode, which allows observation of fresh tissue without any prior fixation or coating, was used with an accelerating voltage of 20kV and vacuum of 0.05 Tort. Stomatal length of 100 stomata were measured using eight leaves, and stomatal density was calculated.

Results and Discussion

Colchicine treatment suppressed cell division. Without colchicine treatment, about 10% of protoplasts developed into colonies and grew to microcalli, while about 2% of protoplasts with the 0.1% colchicine - 3, 6 days treatments. No colony was formed with the 0.1% colchicine - 9 days treatment. 450

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Among calli obtained from colchicine treatments, 52 (0.1% - 3 days) and 31 (0.1% - 6 days) callus lines were analyzed by flow cytometry. It appeared that 29% of the calli (nine of 31 callus lines) from the 0.1% - 6 days treatment had twice the relative nuclear DNA content as the calli derived from untreated protoplasts (Fig. 1). No callus with twice the nuclear DNA content was obtained from the 0.1% - 3 days treatment. No differences in appearance and growth rate were observed between caUi with twice the DNA content and control calli. Eight of nine callus lines with twice the nuclear DNA content formed adventitious buds on the regeneration medium (Table 1) and the buds grew to form shoots. Among eight shoot lines, five lines rooted (Table 2). Compared with 'Jiro', the adventitious bud formation (Table 1) and rooting (Table 2) were lower in the lines with twice the nuclear DNA content, although, the difference was not always significant. By microscopic observation of root tip cells, we have confirmed the doubled chromosome number (2u= 180) of plantlets regenerated from the calli with twice the DNA content (Fig. 2). In some plant species, tissue culture often induces a wide range of somaclonal variation including modification in ploidy level (Creissen and Karp 1985; Ashmore and Shapcott 1989; Nehra et al. 1991; Raja et al. 1992). To confirm this, we investigated 12 plants derived from ordinary protoplast culture and demonstrated that all the 12 plants had original hexaploid chromosome number of 2n=90. The observation, along with no dodecaploid plant regenerated from the 0.1-3% days treatment, could indicate that the dodecaploid plants in this study were true colchiploid, and argues against the possibility of the involvement of somaclonal variation in the origin of dodecaploid plants. Flow cytometric analysis 512

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Fig. 1. Flow cytometric histograms of relative nuclear DNA content of nuclei isolated from Japanese persimmon callus (A: control callus, B: callus from colchicine-treated protoplast). ~

: G0/G 1 phase, ~

: G 2 phase, ~ :

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472 Table 1. Adventitious bud formation from dodecaploid (A-I) and hexaploid (Jiro) callus lines. Data taken from 30 explants after 6 weeks in culture.

Dodecaploid callus lines ~iro' A Adventitious bud formation (%)

B

C

53az 40a 23b

D

E

F

G

H

13b 30ab 10b 20b 20b

I

13b 0c

ZDifferent letters indicate significant difference by chi-square test, P=0.05.

Table 2. Rooting of dodecaploid (A-H) and hexaploid (Jiro) shoots. Data taken after 50 days in culture.

Dodecaploid shoot lines giro' A

B

C

D

E

F

G

H

No. of explants

14

6

8

11

11

6

9

3

3

No. of rooted plants

7

0

2

1

3

2

4

0

0

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revealed that no calli obtained were chimeras because only one G0/G1 peak of nuclear DNA content was observed (Fig. 1). Stomatal guard cell length is a good indicator of ploidy level in some plant species (James et al. 1987; Barrino and Powell 1988; Gupton 1989; Griesbach and Bhat 1990; Fassuliotis and Nelson 1992). In persimmon also, longer stomata length and lower stomata density were observed in dodecaploid plants (Table 3, Fig. 3). Before rooting, dodecaploid shoots grew faster and their leaves were larger than hcxaploid shoots (Fig. 4). However, after acclimatization in the soil, the growth of dodecapioid plants was much slower than the control plants. Decreased growth rate or reduced plant height was also observed in chromosome doubled plants,, such as Rubus species or Eustoma grandiflorum (Gupton 1989; Griesbach and Bhat 1990). Ollitrant-Sammarcelli et al. (1994) showed that kiwifruit plants with high ploidy level had low vigor and dwarfism as compared with control plants. The reduced vigor with increased polyploidy might be used for the production of dwarfing rootstocks of persimmon.

Fig. 2. Chromosome of Japanese persimmon 'Jiro' (A: control plant, 2n=90, B: colchiploid plant, 2n=180).

Table 3. Stomatal characteristics of dodecaploid and hexaploid 'Jiro'. Ploidy level

Stomatal length (ram)

Stomatal density (No./rnm2)

Hexaploid

25.8 _+5.22 z

225 -~65 z

Dodecaploid

39.6 • 10.2

138 _+40

ZMean•

473

Fig. 3. Scanning electron micrographs of stomata (A: control plant, B: colchiploid plant). Bar=-100,um.

Fig. 4. Dodecaploid (fight) and control (left) shoot of 'Jiro'.

References Ashmore SE, Shapcott AS (1989) Cytogenetic studies of Haplopappus gracilis in both callus and suspension cell cultures. Theor Appl Genet 78:249-259 Awoleye F, Duran M, Dolezel J, Novak FJ (1994) Nuclear DNA content and in vitro induced somatic polyploidization cassava (Manihot esculenta Crantz) breeding. Euphytica 76:195-202 Barrino EM, Powell W (1988) Stomatal guard cell length as an indicator of ploidy in microspore-derlved plants of barley. Genome 30:158-160 Creissen GP, Karp A (1985) Karyotypic changes in potato plants regenerated from protoplasts. Plant Cell Tissue Organ Culture 171182 Fassuliotis G, Nelson BV (1992) Regeneration of tetraploid muskmelons from cotyledons and their morphological differences from two diploid muskmelon genotypes. J Amer Soc Hort Sci 117: 863- 866 Goldy RG, Lyrene PM (1984) In vitro colchicine treatment of 4x lblueberries,Vaeeinium sp. J Amer Soc Hort Sci 109:336-338 Griesbach RJ, Bhat RN (1990) Colchicine-induced potyploidy in Eustoma grandiflorum. HortScience 25:1284-1286

Gupton CL (1989) Production of non-chimeral colchiploids in Rubus species by tissue culture. Euphytica 44:133-135 James DJ, Mackenize KAD, Malhotra SB (1987) The induction of hexaploidy in cherry rootstocks using in vitro regeneration techniques. Theor Appl Genet 73:589-594 Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126:105-110 Kitagawa H, Glucina PG (1984) Persimmon culture in New Zealand. Sci Inf Publ Cent, Wellington Namikawa I (1930) On the chromosomes of the persimmon. Transactions of the Tottorl Soc Agri Sci 2:145-148 (in Japanese) Namikawa I, Higashi M (1928) On the number of chromosomes in D. kaki Linn. and D. lotus Linn. Bot Mag 42:436-438 Nehra SN, Kartha KK, Stushnoff C (1991) Nuclear DNA content and isozyme variation in relation to morphogenic potential of strawberry (Fragaria x ananassa) callus cultures. Can J Bot 69:239-244 Ollitrault-Sammarcelli F, Legave JM, Michaux-Ferriere N,Hirsch AM (1994) Use of flow cytometry for rapid detemaination of ploidy level in the genus Actinidia. Scientia Horticulturae 57:303-313