Plant Cell Reports (1983) 2:314-317
PlantCell Reports
© Springer-Verlag 1983
Plant Regeneration from Protoplast-Derived Tissues of Linum usitatissimum L. (Flax) M. N. Barakat* and E. C. Cocking Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received June 28, 1983 / November 9, 1983 - Communicated by J. Potrykus
ABSTRACT Protoplasts were isolated enzymatically from seedling roots, hypocotyls and cotyledons of Linum usitatissimum L. which divided to form callus. Plant regeneration was obtained from protoplast-derived tissues of root and cotyledon, but only rhizogenesis was observed in the case of protoplasts derived from hypocotyls. The a b i l i t y to i s o l a t e , c u l t u r e , and regenerate plants from root and cotyledon protoplasts of Linum usitatissimum L. is discussed in r e l a t i o n to future attempts to produce somatic hybrids between Linum species. Abbreviations BAP 2,4-D IAA NAA CPW
6-benzylaminopurine 2,4-dichlorophenoxyacetic acid 3,indoleacetic acid ~-naphthaleneacetic acid Cell and protoplast wash solution INTRODUCTION
Linum usitatissimum L. (Flax) is an important source of natural f i b r e s , and i t s seeds contain o i l and s i g n i f i c a n t amounts of protein (Harapiak 1974). The leading countries in Flax seed production in order of importance are I n d i a , Argentina, USSR, Canada and the United States. Several of the wild species of Linum possess many agronomically valuable genes suchas resistance to diseases (such as the f l a x rust Melamspora l i n i ) , and to drought, winter hardiness, e t c . , besides an unexplored reservoir of genes for o i l q u a l i t y (Plesser 1966; Yermanos et al. 1966) which may be of great value i f transferred to f l a x (Seetharam 1972). Crosses between species with 15 chromosomes (2n=30) have been successful (Tammes 1928; G i l l and Yermanos 1967a; Bari and Godward 1970; Seetharam 1972; Wicks and Hammond 1978). Crosses between Linum species with 9 chromosomes (2n=18) are also f r e q u e n t l y successful, but crosses between nine-chromosome species and Linum usitatissimum (2n=30), or any other species with f i f t e e n chromosomes (2n=30) f a i l e d to have produced seeds ( G i l l and Yermanos 1967b; Bari and Godward 1970; Seetharam 1972). Unfortunately, c h a r a c t e r i s t i c s of most i n t e r e s t , that are not found in Linum usitatissimum are present only in some of the nine-chromosome species.
Protoplast fusion may be a potential method of achieving hybrids that are otherwise unobtainable (Cocking et al. 1981). This technique was i n i t i a l l y used to produce i n t e r s p e c i f i c hybrids between sexually compatible species of Nicotiana (Carlson et al. 1972; Smith et al. 1976) and Petunia (Power et al. 1976). This report summarises the conditions f o r i s o l a t i o n , culture and regeneration of plants from various protoplast systems suitable for the genetic manipulation of somatic c e l l s of Linum usitatissimum L. MATERIALS AND i.IETHODS Plant material - seeds of Linum usitatissimum cv Precederia were obtained from t ~ Department of Crop Science, U n i v e r s i t y of Alexandria, Alexandria, Egypt. Seeds were s t e r i l i s e d in I0% v/v Domestos bleach (Lever Bros. U.K.) f o r 30 min and washed with 6 changes of s t e r i l e tap water. Seeds were germinated on agar s o l i d i f i e d (0.8% w/v; Sigma) Murashige and Skoog (1962) based medium (MS) containing 3% w/v sucrose, but lacking growth regulators, and incubated in the dark at 23±2oc. Protoplast i s o l a t i o n - young cotyledons and hypocotyls were excised a f t e r 7 days; roots were excised a f t e r 3 days. Cotyledons were cut into approximately l mm wide s t r i p s , however, hypocotyls and roots were cut l o n g i t u d i n a l l y . The tissues for d i f f e r e n t explants were plasmolysed in CPW solution containing 13% w/v mannitol (Frearson et al. 1973) for two hoursythen incubated in an enzyme solution Of 2% w/v Rhozyme HP 150 (R~hm and Haas L t d . , Philadelphia, U.S.A.), 4% w/v Meicelase (Meiji Seika Kaisha L t d . , Tokyo, Japan) and 0.3% w/v macerozyme (Yakult Biochemicals L t d . , Nishinomiya, Japan) in CPW salts solution with 13% w/v mannitol. Enzyme incubation was for 16 h at 25°C on a rotary shaker (30-40 rpm). Protoplasts were released by gentle squeezing of the d i f f e r e n t explants and f i l t e r e d through a nylon sieve of 64 um pore size. Cotyledon protoplasts were pelleted by c e n t r i f u g a t i o n at lO0 x g, lO min and washed by c e n t r i f u g a t i o n using three changes of CPW 13% mannitol solution. Hypocotyl protopiasts were pelleted by c e n t r i f u g a t i o n at lO0 x g,5 min; in t h i s step most of the protoplasts were f l o a t i n g on the surface of enzyme solution because protoplasts were h i g h l y vacuolated. Protoplasts that had pelleted were p u r i f i e d by f l o t a t i o n in CPW 21% w/v sucrose (lO0 x g f o r lO min). Root protoplasts were pelleted by c e n t r i f u g a t i o n at lO0 x g
* o n leave from Department of Crop Science, University of Alexandria, Egypt
315 f o r 5 min and then p u r i f i e d by f l o t a t i o n in CPW 21% w/v sucrose (lO0 x g, for lO min). Floating protoplasts were washed twice with CPW 13% w/v mannitol, resuspended in the c u l t u r e medium, and counted. Protoplast culture - Protoplasts were cultured in the dark and in d i f f u s e l i g h t (700 lux 23±2°C) in 54 x 14 mm Nunclon dishes (A/S Nunc, Kamstrup, DKL4000 Roskilde, Denmark) e i t h e r in :- 4.0 ml l i q u i d medium, or 1.0 ml l i q u i d medium over 4.0 ml agar s o l i d i f i e d (0.5% w/v; Sigma) medium, or l i q u i d over agar, but with the protoplasts plated onto a s t e r i l e f i l t e r paper l a i d on the agar surface (Santos et al. 1980). The f o l l o w i n g media were used; MSP-I (Murashige and Skoog, 1962 medium modified by adding 2 mg/L NAA and 0.5 mg/L BAP) with 9% w/v mannitol. This was d i l u t e d with MSP-I containing 6% w/v mannitol, 3% w/v mannitol and f i n a l l y MSP-I with no mannitol; MB5P (Gamborg and Shyluk 1976). This was d i l u t e d with MB5, the same components as in MB5P except f o r hormones and sugar (0.I mg/L zeatin, 0.2 mg/L NAA, 0.I mg/L 2,4-D, 20 g/L sucrose and I0 g/L glucose) in the proportion of 2:1, I : I , and 1:2 v / v ; KSP (Kao, 1977) containing 0.2 mg/L 2,4-D, 0.5 mg/L zeatin and 1.0 mg/L NAA. This was d i l u t e d with K8 (Kao, 1977) containing 0.I mg/L 2,4-D, 0.2 mg/L zeatin and 1.0 mg/L NAA. 2% w/v sucrose and I% w/v glucose, 2:1, I : I , and 1:2 v / v ; and KM8P:KM8 (2:1, v : v , Kao and Michayluk 1975). This was d i l u t e d in the proportion of I : I , 1:2, and 0:I v/v of KM8P:KMS. The p l a t i n g density of protoplasts was adjusted to 0.25, 0.5 and 1.0 x 105/ml . The osmotic concentration of the culture medium was lowered r e g u l a r l y at weekly i n t e r v a l s . The p l a t i n g e f f i c i e n c y (number of d i v i d i n g protoplasts expressed as a percentage of the t o t a l protoplast population) was estimated a f t e r 7 days from three experiments, each experiment was replicated three times. Regeneration media - Colonies derived from cotyledon protoplasts were transferred, a f t e r 2 weeks, to regeneration media (Table 2). However, colonies derived from root and hypocotyl protoplasts were transferred, a f t e r 4 weeks, to the regeneration media. Cultures f o r plant regeneration were kept under continuous fluorescent l i g h t 800 lux at 23±2°C. Regenerated shoot t i p s (with t h r e e - four leaves) were transferred to hormone-free MS agar medium, and also to h a l f strength of B5 medium (Gamborg et al. 1968) with 0.2 mg/L IAA for root i n d u c t i o n , and p l a n t l e t s potted in s o i l - l e s s compost, hardened o f f and maintained in the greenhouse. RESULTS AND DISCUSSION Previously plant regeneration from protoplastderived tissues of Linum species has not been obtained. Hypocotyl protoplast-derived callus of Linum usitatissimum has, however, been reported to regenerate roots (Gamborg and Shyluk 1976). This is the f i r s t report of plant regeneration from protoplasts of Linum usitatissimum. I t was possible to release protoplasts from a l l sources of plant material investigated. Yields ranged from 25 x 105/g fresh weight (hypocotyl) to 21 x 106/g fresh weight (cotyledon). The protoplasts varied in size depending on the source of plant material. The protoplast population from the three explants of f l a x consisted of unvacuolated protoplasts in the case of cotyledon and root explants, and vacuolated protoplasts in the case of hypocotyl explants. So consequently hypocotyl protoplasts had
a strong tendency to f l o a t during the f i r s t stage of c e n t r i f u g a t i o n . F i r s t d i v i s i o n s occurred a f t e r 48 h of culture in the case of cotyledon and hypocotyl protoplasts, and a f t e r 72 h of c u l t u r e in the case of root protoplasts. Division was best at 5 x 104/ml for cotyledon and hypocotyl protoplasts and at 2.5 x 104/ml for root protoplasts. The p l a t i n g e f f i c i e n c y was greatest for protoplasts from root and from the other types of plant material in Kao and Michayluk medium under l i g h t condition (Table l ) . Of plant meristems those from roots have the highest rate of d i v i s i o n (Clowes 1976); protoplasts from root apices might therefore be expected to have a high d i v i s i o n p o t e n t i a l . Studies with Linum emphasise the usefulness of the rich media, o r i g i n a l l y developed by Kao and Michavluk (1975) and Kao (1977] to stimulate protoplast d i v i s i o n of several species (Kao et al. 1980; Johnson et al. 1981; Berry et al. 1982; Lu et al. 1982; Xu et al. 1982c; Ahuja et al. 1983 and Lu et al. 1983a, 1983b). The f i n d i n g that protoplast d i v i s i o n is improved when in d i f f u s e l i g h t is in agreement with the e a r l i e r r e s u l t s of Gamborg and Shyluk (1976). Table I . Effect of culture conditions and d i f f e r e n t media on p l a t i n g e f f i c i e n c y for d i f f e r e n t explants. Condition
Media MSP-I MB5P
Dark
Light
Root
Hypocotyl
Cotyledon
12.4 _+ 0.7 I18.2 + 1.0 25.6 _+ 1.2 24.8 ± 0.6 22.9 ± 1.2 20.2 ± 0.5
32.6 ± 2.7 29.6 ± I . I 27.2 ± 1.3 KM8P:KM8 33.2 ± 1.3 23.7 ± 1.0 22.5 _+ 0.8
K8P
MSP-I
16.9 ± 0.5 2 6 . 0 ± 1 . 2
27.6 ± 0.8
MB5P
26.5 ± 0.4 2 5 . 6 ± 1 . 2
26.7 _+ 1.7
K8P
38.4 ± 1.5 3 0 . 2 ± 0 . 8
32.4 ± 0.7
31.5±0.7
35.0 ± 1.7
KM8P:KM8 38.4 ± I . I
The protoplast-derived colonies became brown and deteriorated a f t e r 8 days in l i q u i d medium. The amount of medium used f o r d i l u t i o n is an essential f a c t o r in solving the problem of browning of colonies derived from protoplasts in the case of c u l t u r e using l i q u i d over agar. I t was observed that d i l u t i o n with 5 drops of medium was better than with 1 ml of medium. Cell colonies derived from cotyledon protoplasts were transferred onto d i f f e r e n t a t i o n media f o r regeneration a f t e r 2 weeks and did not need any d i l u t i o n . Cotyledon protoplasts on f i l t e r paper continued to divide and gave rise to large colonies. Cell colonies could be seen under a stereo microscope w i t h i n 7 days of p l a t i n g , and became v i s i b l e to the naked eye a f t e r I0 days of c u l t u r e . Although the best p l a t i n g e f f i c i e n c y was obtained using Kao and Michayluk medium, the growth of colonies was more rapid in MB5P l i q u i d over agar (Fig 3). Colony formation from root and hypocotyl protoplasts on MSP-I medium, using f i l t e r paper support was r a r e l y achieved. The problem of the browning colonies was solved e i t h e r by c u l t u r i n g protoplasts in l i q u i d medium and then a f t e r 7 days t r a n s f e r r i n g the colonies into d i l u t e d l i q u i d over agar medium, or by c u l t u r i n g protop!asts immediately e i t h e r on l i q u i d over agar, or p l a t i n g protoplasts onto a s t e r i l e f i l t e r paper l a i d on the agar surface. A f i l t e r paper substratum was i n i t i a l l y used in the culture of Medicago sativa protoplasts to reduce browning (Santos et a l , 1980), but t h i s has since been
316 rendered unnecessary by using seedling plant m a t e r i a l , (Lu et a l . 1982). Gamborg and Shyluk (1976) found that some Linum hypocotyl protoplasts divided once or twice, b u t ~ d e t e r i o r a t e d a f t e r about I0 days of culture. When the dishes were gently shaken a f t e r the i n i t i a l c e l l d i v i s i o n s , the droplets mixed and the cells became attached to the bottom surface of the petri dish and continued to grow. At the same time fresh medium was added. Regeneration of Protoplast-derived tissuesThe response of d i f f e r e n t protoplast-derived c a l l i to various media is summarised in (Table 2). In most of the media, the colonies grew into compact c a l l i and produced masses of green nodules. The nodules f a i l e d to regenerate into shoots a f t e r three subcultures on fresh aliquots of the same medium from which they had developed. The modified Murashige and Skoog (1962) agar medium containing BAP 1.13 mg/L and NAA 0.02 mg/L was the best f o r shoot regeneration from root and cotyledon protoplast-derived tissues of Linum. I t was suitable for shoot regeneration in s t e m ~ n t s of Brassica napus (Kartha et a l , 1974) and suitable f o r s-TT6-6-lTregeneration in stem and callus explants of Linum usitatissimum (Murray et a l , 1977). Root ~tion was induced when shoot tips with 3-4 leaves were transferred to MS based agar, without phytohormones, and h a l f strength of B5 based agar with 0.2 mg/L IAA (Figs. l - 5 ) . Previously i t has been emphasised that the use of protoplasts isolated from seedling roots (Xu et a l . 1981, 1982) and from seedling cotyledons (Lu et a l . 1982) could be p a r t i c u l a r l y advantageous when somatic hybridization assessments are being undertaken. The use of germinating seeds overcomes the d i f f i c u l t i e s often encountered with the production of l e a f material, and enables an adequate supply of experimental material to be produced rapidly with the minimum of resources. I t has also been suggested for somatic hybridization assessments that protoplasts from roots and e t i o l a t e d cotyledons could be used, instead of cell suspension protoplasts, f o r fusion with green cotyledon l e a f protoplasts or l e a f mesophyll protoplasts (Lu et a l , 1982). Heterokaryons can be i d e n t i f i e d and isolated manually (Patnaik et a l , 1982). Moreover, the y i e l d of cotyledon protoplasts is also often adequate for genetic transformation studies (Cocking et a l , 1981). These present studies on plant regeneration from root and cotyledon protoplasts of Linum usitatissimum w i l l provide an adequate cultural for such genetic manipulation assessments. The culture c a p a b i l i t i e s of the other species of Linum are also currently being investigated.
Fig. I . Freshly isolated protoplasts. a-Root~ XI50o b-Hypocotyl~XlO0. C-Cotyledon,X125. Fig. 2. Cell colonies derived from hypocotyl protoplasts a f t e r 7 days of culture on l i q u i d medium. X300. Fig. 3. Cell colonies derived from cotyledon protoplasts a f t e r 15 days of culture on solid medium: 5 cm diameter dish. a-culture in KM8P:KM8 medium, b-culture in MB5P medium. Fig. 4. P r o l i f i c shoot regeneration from callus derived from cotyledon protoplasts.
Table 2. Response of d i f f e r e n t explant protoplast-derived callus to various media. Medium
Protopl ast-derived callus
Composition
Root MSP-I MSP-3 MSD-3 MSD-4 MSF MSZ B5-1 B5-2
Hypocotyl Cotyledon
Murashige and Skoog 1962 (2.0 mg/L NAA and 0.5 mg/L BAP) Murashige and Skoog 1962 (0.I mg/L NAA and 0.5 mg/L BAP)
A,R
A,R
A,R
+,S
Murashige and Skoog 1962 (2.0 mg/L IAA and 1.0 mg/L BAP) Murashige and Skoog 1962 (0.05 mg/L N/~A and 0.5 mg/L BAP)
A,R,+
+,R A,R+
+,R A,R,+
A,R,+
A,R,+
A,R,+
++,S
+,R
++
Murashige and Skoog 1962 (0.02 mg/L NR~Aand 1.13 mg/L BAP) Murashige and Skoog 1962 ( I . 0 mg/L Zeatin) Gamborg et a l , 1968 (0.02 mg/L NAA and 2.0 mg/L BAP) Gamborg et a l , 1968 (0.I mg/L NAA and 0.5 mg/L BAP)
R= Roots S= Shoots A= Abundant callus ++ = Large green nodular callus.
+ = small
,S
++
++
++
+
+
+
+ +
+
green nodular callus
317 Kao KN (1977) Chromosomal behaviour in somatic hybrids of soybean - Nicotiana glauca, Mol. Gen. Genet. 150: 225-230. Kao KN, Michayluk MR (1980) Plant regeneration from mesophyll protoplasts of a l f a l f a . Z. Pflanzenphysiol. 96: 135-141. Kartha KK, Gamborg OL, Constabel F (1974) In v i t r o plant formation from stem explants of rape (Brassica napus cv. Zephyr). Physiol. Plant
3i7..~2G. Fig. 5. Plant regeneration from cotyledon protoplasts of f l a x , Linum usitatissimum, ACKNOWLEDGEMENTS M. Barakat was supported by an Egyptian Education Bureau Scholarship. Thanks are due to Dr. F.S. EI-Nakhlaway, Alexandria, Egypt) for seed samples. The photographic assistance of Mr. B.V. Case is g r a t e f u l l y acknowledged. REFERENCES Ahuja PS, Lu DY, Cocking EC, Davey MR (1983) An Assessment of the cultural c a p a b i l i t i e s of Trifolium repens L. (White Clover) and Onobrychis v i c i i f o l i a Scop. (sainfoin) mesophyll protoplasts. Plant Cell Reports (in press). Bari G, Godward MBE (1970) I n t e r s p e c i f i c crosses in Linum. Euphytica 19: 443.446. Berry SF, Lu DY, Pental D, Cocking EC (1982) Regeneration of plants from protopiasts of Lactuca sativa L. Z. Pflanzenphysiol. I08: 31-38. Carlson PS, Smith HH, Dearing RD (1972) Parasexual i n t e r s p e c i f i c plant hybridization. Proc. Natl. Acad. Sci. USA 69: 2292-2294. Clowes FAL (1976) The root apex. In: Yeoman MM (ed) Cell Division in Higher Plants, Academic Press, pp 253-284. Cocking EC, Davey MR, Pental D, Power JB (1981) Aspects of plant genetic manipulation. Nature 293: 265-270. Frearson EM, Power JB, Cocking EC (1973) The isol a t i o n , culture and regeneration of Petunia leaf protoplasts. Dev. Biol. 33: 130-137. Gamborg OL, M i l l e r RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root c e l l s . Exp. Cell Res. 50: 151-158. Gamborg OL, Shyluk JP (1976) Tissue culture, protoplasts and morphogenesis in f l a x . Bot. Gaz. 137: 301-306. G i l l KS, Yermanos DM (1967a) Cytogenetic studies on the genus Linum. I. Hybrids among taxa with 15 as the hap~chromosome number. Crop Sci. 7: 623-627. G i l l KS, Yermanos DM (1967b) Cytogenetic studies on the genus Linum. I I . Hybrids among taxa with nine as the haploid chromosome number. Crop Sci. 7: 627-631. Harapiak JT (1974) Oilseed and pulse crops in Western Canada. Western Crops F e r t i l i z e r , Calgary. Johnson LB, S t u t e v i l l e DL, Higgins RK, Skinner DZ (1981) Regeneration of a l f a l f a plants from protoplasts of selected Regen S clones. Plant Sci. Lett. 20: 297-304. Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana c e l l s and protoplasts at very low population density in l i q u i d media. Planta 126: 105-110.
Lu DY, Pental D, Cocking EC (1982) Plant regeneration from seedling cotyledon protoplasts. Z. Pflanzenphysiol. 107: 59-63. Lu DY, Cooper-Bland S, Pental D, Cocking EC, Davey MR (1983a) Isolation and sustained d i v i s i o n of protoplasts from cotyledons and seedlings and immature seeds of Glycine max, soybean. Z. Pflanzenphysiol. I ] ~ - ~ . Lu DY, Davey MR, Cocking EC (1983b) A comparison of the cultural behaviour of protoplasts from leaves, cotyledons and roots of Medicago sativa L. Plant Sci. Lett. (in press). Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. Murray BE, Handyside RJ, Keller WA (1977) In v i t r o regeneration of shoots on stem explants o ~ p l o i d and diploid f l a x (Linum usitatissimum). Can. J. Genet. Cytol. 19: ~ 8 ~ Patnaik G, Cocking EC, Hamill J, Pental D (1982) A simple procedure for the manual i s o l a t i o n and i d e n t i f i c a t i o n of plant heterokaryons. Plant Sci. Lett. 24: 105-110. Plesser AG (1966) The v a r i a t i o n in f a t t y acid composition of the seed of Linum species. Can. J. Genet. Cytol. 8: 328-335. Power JB, Frearson EM, Hayward C, George D, Evans PK, Berry SF, Cocking EC (1976) Somatic hybridization of Petunia hybrida and P. parodii. Nature 263: 5 0 0 - ~ Santos AVP dos, Outka DE, Cocking EC, Davey MR (1980) Organogenesis and somatic embryogenesis in tissues derived from l e a f protoplasts and l e a f explants of Medicago sativa. Z. Pflanzenphysiol. 99: 261-270. Seetharam A (1972) I n t e r s p e c i f i c hybridization in Linum. Euphytica 21: 489-495. SmithHH, Kao KN, Combatti NC (1976) I n t e r s p e c i f i c hybridization by protoplast fusion in Nicotiana. J. Hered. 67: 123-128. Tammes T (1928) The genetics of the genus Linum. Bibliogr. Genet. 4: 1-36. Wicks ZW,III, Hammond JJ (1978) Screening Linum species for new sources of genes resistant to melamspora L i n i . Crop Sci. 18: 7-I0. Xu Z-H, Davey MR, Cocking EC (1981) Isolation and sustained d i v i s i o n of Phaseolus aureus (mung bean) root protoplasts. Z. Pflanzenphysiol. I04: 289298. Xu Z-H, Davey MR, Cocking EC (1982a) Callus formation from root protoplasts of Glycine max (soybean). Plant Sci.Lett. 24.7--i77111-I15. Xu Z-H, Davey MR, Cocking EC (1982b) Plant regeneration from root protoplasts of Brassica. Plant Sci. Lett. 24: ll7-121. Xu Z-H, Davey MR, Cocking EC (1982c) Organogenesis from protoplasts of forage legumes Medicago sativa and Trigonella foenum-graecum. Z. Pflanzenphysiol. I07: 231-235. Yermanos DM, Beard BH, G i l l KS, Anderson MP (1966) Fatty acid composition of seed o i l of wild species of Linum. Agron. J. 58: 30-32.
PlantCell Reports
© Springer-Verlag 1983
Plant Regeneration from Protoplast-Derived Tissues of Linum usitatissimum L. (Flax) M. N. Barakat* and E. C. Cocking Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, UK Received June 28, 1983 / November 9, 1983 - Communicated by J. Potrykus
ABSTRACT Protoplasts were isolated enzymatically from seedling roots, hypocotyls and cotyledons of Linum usitatissimum L. which divided to form callus. Plant regeneration was obtained from protoplast-derived tissues of root and cotyledon, but only rhizogenesis was observed in the case of protoplasts derived from hypocotyls. The a b i l i t y to i s o l a t e , c u l t u r e , and regenerate plants from root and cotyledon protoplasts of Linum usitatissimum L. is discussed in r e l a t i o n to future attempts to produce somatic hybrids between Linum species. Abbreviations BAP 2,4-D IAA NAA CPW
6-benzylaminopurine 2,4-dichlorophenoxyacetic acid 3,indoleacetic acid ~-naphthaleneacetic acid Cell and protoplast wash solution INTRODUCTION
Linum usitatissimum L. (Flax) is an important source of natural f i b r e s , and i t s seeds contain o i l and s i g n i f i c a n t amounts of protein (Harapiak 1974). The leading countries in Flax seed production in order of importance are I n d i a , Argentina, USSR, Canada and the United States. Several of the wild species of Linum possess many agronomically valuable genes suchas resistance to diseases (such as the f l a x rust Melamspora l i n i ) , and to drought, winter hardiness, e t c . , besides an unexplored reservoir of genes for o i l q u a l i t y (Plesser 1966; Yermanos et al. 1966) which may be of great value i f transferred to f l a x (Seetharam 1972). Crosses between species with 15 chromosomes (2n=30) have been successful (Tammes 1928; G i l l and Yermanos 1967a; Bari and Godward 1970; Seetharam 1972; Wicks and Hammond 1978). Crosses between Linum species with 9 chromosomes (2n=18) are also f r e q u e n t l y successful, but crosses between nine-chromosome species and Linum usitatissimum (2n=30), or any other species with f i f t e e n chromosomes (2n=30) f a i l e d to have produced seeds ( G i l l and Yermanos 1967b; Bari and Godward 1970; Seetharam 1972). Unfortunately, c h a r a c t e r i s t i c s of most i n t e r e s t , that are not found in Linum usitatissimum are present only in some of the nine-chromosome species.
Protoplast fusion may be a potential method of achieving hybrids that are otherwise unobtainable (Cocking et al. 1981). This technique was i n i t i a l l y used to produce i n t e r s p e c i f i c hybrids between sexually compatible species of Nicotiana (Carlson et al. 1972; Smith et al. 1976) and Petunia (Power et al. 1976). This report summarises the conditions f o r i s o l a t i o n , culture and regeneration of plants from various protoplast systems suitable for the genetic manipulation of somatic c e l l s of Linum usitatissimum L. MATERIALS AND i.IETHODS Plant material - seeds of Linum usitatissimum cv Precederia were obtained from t ~ Department of Crop Science, U n i v e r s i t y of Alexandria, Alexandria, Egypt. Seeds were s t e r i l i s e d in I0% v/v Domestos bleach (Lever Bros. U.K.) f o r 30 min and washed with 6 changes of s t e r i l e tap water. Seeds were germinated on agar s o l i d i f i e d (0.8% w/v; Sigma) Murashige and Skoog (1962) based medium (MS) containing 3% w/v sucrose, but lacking growth regulators, and incubated in the dark at 23±2oc. Protoplast i s o l a t i o n - young cotyledons and hypocotyls were excised a f t e r 7 days; roots were excised a f t e r 3 days. Cotyledons were cut into approximately l mm wide s t r i p s , however, hypocotyls and roots were cut l o n g i t u d i n a l l y . The tissues for d i f f e r e n t explants were plasmolysed in CPW solution containing 13% w/v mannitol (Frearson et al. 1973) for two hoursythen incubated in an enzyme solution Of 2% w/v Rhozyme HP 150 (R~hm and Haas L t d . , Philadelphia, U.S.A.), 4% w/v Meicelase (Meiji Seika Kaisha L t d . , Tokyo, Japan) and 0.3% w/v macerozyme (Yakult Biochemicals L t d . , Nishinomiya, Japan) in CPW salts solution with 13% w/v mannitol. Enzyme incubation was for 16 h at 25°C on a rotary shaker (30-40 rpm). Protoplasts were released by gentle squeezing of the d i f f e r e n t explants and f i l t e r e d through a nylon sieve of 64 um pore size. Cotyledon protoplasts were pelleted by c e n t r i f u g a t i o n at lO0 x g, lO min and washed by c e n t r i f u g a t i o n using three changes of CPW 13% mannitol solution. Hypocotyl protopiasts were pelleted by c e n t r i f u g a t i o n at lO0 x g,5 min; in t h i s step most of the protoplasts were f l o a t i n g on the surface of enzyme solution because protoplasts were h i g h l y vacuolated. Protoplasts that had pelleted were p u r i f i e d by f l o t a t i o n in CPW 21% w/v sucrose (lO0 x g f o r lO min). Root protoplasts were pelleted by c e n t r i f u g a t i o n at lO0 x g
* o n leave from Department of Crop Science, University of Alexandria, Egypt
315 f o r 5 min and then p u r i f i e d by f l o t a t i o n in CPW 21% w/v sucrose (lO0 x g, for lO min). Floating protoplasts were washed twice with CPW 13% w/v mannitol, resuspended in the c u l t u r e medium, and counted. Protoplast culture - Protoplasts were cultured in the dark and in d i f f u s e l i g h t (700 lux 23±2°C) in 54 x 14 mm Nunclon dishes (A/S Nunc, Kamstrup, DKL4000 Roskilde, Denmark) e i t h e r in :- 4.0 ml l i q u i d medium, or 1.0 ml l i q u i d medium over 4.0 ml agar s o l i d i f i e d (0.5% w/v; Sigma) medium, or l i q u i d over agar, but with the protoplasts plated onto a s t e r i l e f i l t e r paper l a i d on the agar surface (Santos et al. 1980). The f o l l o w i n g media were used; MSP-I (Murashige and Skoog, 1962 medium modified by adding 2 mg/L NAA and 0.5 mg/L BAP) with 9% w/v mannitol. This was d i l u t e d with MSP-I containing 6% w/v mannitol, 3% w/v mannitol and f i n a l l y MSP-I with no mannitol; MB5P (Gamborg and Shyluk 1976). This was d i l u t e d with MB5, the same components as in MB5P except f o r hormones and sugar (0.I mg/L zeatin, 0.2 mg/L NAA, 0.I mg/L 2,4-D, 20 g/L sucrose and I0 g/L glucose) in the proportion of 2:1, I : I , and 1:2 v / v ; KSP (Kao, 1977) containing 0.2 mg/L 2,4-D, 0.5 mg/L zeatin and 1.0 mg/L NAA. This was d i l u t e d with K8 (Kao, 1977) containing 0.I mg/L 2,4-D, 0.2 mg/L zeatin and 1.0 mg/L NAA. 2% w/v sucrose and I% w/v glucose, 2:1, I : I , and 1:2 v / v ; and KM8P:KM8 (2:1, v : v , Kao and Michayluk 1975). This was d i l u t e d in the proportion of I : I , 1:2, and 0:I v/v of KM8P:KMS. The p l a t i n g density of protoplasts was adjusted to 0.25, 0.5 and 1.0 x 105/ml . The osmotic concentration of the culture medium was lowered r e g u l a r l y at weekly i n t e r v a l s . The p l a t i n g e f f i c i e n c y (number of d i v i d i n g protoplasts expressed as a percentage of the t o t a l protoplast population) was estimated a f t e r 7 days from three experiments, each experiment was replicated three times. Regeneration media - Colonies derived from cotyledon protoplasts were transferred, a f t e r 2 weeks, to regeneration media (Table 2). However, colonies derived from root and hypocotyl protoplasts were transferred, a f t e r 4 weeks, to the regeneration media. Cultures f o r plant regeneration were kept under continuous fluorescent l i g h t 800 lux at 23±2°C. Regenerated shoot t i p s (with t h r e e - four leaves) were transferred to hormone-free MS agar medium, and also to h a l f strength of B5 medium (Gamborg et al. 1968) with 0.2 mg/L IAA for root i n d u c t i o n , and p l a n t l e t s potted in s o i l - l e s s compost, hardened o f f and maintained in the greenhouse. RESULTS AND DISCUSSION Previously plant regeneration from protoplastderived tissues of Linum species has not been obtained. Hypocotyl protoplast-derived callus of Linum usitatissimum has, however, been reported to regenerate roots (Gamborg and Shyluk 1976). This is the f i r s t report of plant regeneration from protoplasts of Linum usitatissimum. I t was possible to release protoplasts from a l l sources of plant material investigated. Yields ranged from 25 x 105/g fresh weight (hypocotyl) to 21 x 106/g fresh weight (cotyledon). The protoplasts varied in size depending on the source of plant material. The protoplast population from the three explants of f l a x consisted of unvacuolated protoplasts in the case of cotyledon and root explants, and vacuolated protoplasts in the case of hypocotyl explants. So consequently hypocotyl protoplasts had
a strong tendency to f l o a t during the f i r s t stage of c e n t r i f u g a t i o n . F i r s t d i v i s i o n s occurred a f t e r 48 h of culture in the case of cotyledon and hypocotyl protoplasts, and a f t e r 72 h of c u l t u r e in the case of root protoplasts. Division was best at 5 x 104/ml for cotyledon and hypocotyl protoplasts and at 2.5 x 104/ml for root protoplasts. The p l a t i n g e f f i c i e n c y was greatest for protoplasts from root and from the other types of plant material in Kao and Michayluk medium under l i g h t condition (Table l ) . Of plant meristems those from roots have the highest rate of d i v i s i o n (Clowes 1976); protoplasts from root apices might therefore be expected to have a high d i v i s i o n p o t e n t i a l . Studies with Linum emphasise the usefulness of the rich media, o r i g i n a l l y developed by Kao and Michavluk (1975) and Kao (1977] to stimulate protoplast d i v i s i o n of several species (Kao et al. 1980; Johnson et al. 1981; Berry et al. 1982; Lu et al. 1982; Xu et al. 1982c; Ahuja et al. 1983 and Lu et al. 1983a, 1983b). The f i n d i n g that protoplast d i v i s i o n is improved when in d i f f u s e l i g h t is in agreement with the e a r l i e r r e s u l t s of Gamborg and Shyluk (1976). Table I . Effect of culture conditions and d i f f e r e n t media on p l a t i n g e f f i c i e n c y for d i f f e r e n t explants. Condition
Media MSP-I MB5P
Dark
Light
Root
Hypocotyl
Cotyledon
12.4 _+ 0.7 I18.2 + 1.0 25.6 _+ 1.2 24.8 ± 0.6 22.9 ± 1.2 20.2 ± 0.5
32.6 ± 2.7 29.6 ± I . I 27.2 ± 1.3 KM8P:KM8 33.2 ± 1.3 23.7 ± 1.0 22.5 _+ 0.8
K8P
MSP-I
16.9 ± 0.5 2 6 . 0 ± 1 . 2
27.6 ± 0.8
MB5P
26.5 ± 0.4 2 5 . 6 ± 1 . 2
26.7 _+ 1.7
K8P
38.4 ± 1.5 3 0 . 2 ± 0 . 8
32.4 ± 0.7
31.5±0.7
35.0 ± 1.7
KM8P:KM8 38.4 ± I . I
The protoplast-derived colonies became brown and deteriorated a f t e r 8 days in l i q u i d medium. The amount of medium used f o r d i l u t i o n is an essential f a c t o r in solving the problem of browning of colonies derived from protoplasts in the case of c u l t u r e using l i q u i d over agar. I t was observed that d i l u t i o n with 5 drops of medium was better than with 1 ml of medium. Cell colonies derived from cotyledon protoplasts were transferred onto d i f f e r e n t a t i o n media f o r regeneration a f t e r 2 weeks and did not need any d i l u t i o n . Cotyledon protoplasts on f i l t e r paper continued to divide and gave rise to large colonies. Cell colonies could be seen under a stereo microscope w i t h i n 7 days of p l a t i n g , and became v i s i b l e to the naked eye a f t e r I0 days of c u l t u r e . Although the best p l a t i n g e f f i c i e n c y was obtained using Kao and Michayluk medium, the growth of colonies was more rapid in MB5P l i q u i d over agar (Fig 3). Colony formation from root and hypocotyl protoplasts on MSP-I medium, using f i l t e r paper support was r a r e l y achieved. The problem of the browning colonies was solved e i t h e r by c u l t u r i n g protoplasts in l i q u i d medium and then a f t e r 7 days t r a n s f e r r i n g the colonies into d i l u t e d l i q u i d over agar medium, or by c u l t u r i n g protop!asts immediately e i t h e r on l i q u i d over agar, or p l a t i n g protoplasts onto a s t e r i l e f i l t e r paper l a i d on the agar surface. A f i l t e r paper substratum was i n i t i a l l y used in the culture of Medicago sativa protoplasts to reduce browning (Santos et a l , 1980), but t h i s has since been
316 rendered unnecessary by using seedling plant m a t e r i a l , (Lu et a l . 1982). Gamborg and Shyluk (1976) found that some Linum hypocotyl protoplasts divided once or twice, b u t ~ d e t e r i o r a t e d a f t e r about I0 days of culture. When the dishes were gently shaken a f t e r the i n i t i a l c e l l d i v i s i o n s , the droplets mixed and the cells became attached to the bottom surface of the petri dish and continued to grow. At the same time fresh medium was added. Regeneration of Protoplast-derived tissuesThe response of d i f f e r e n t protoplast-derived c a l l i to various media is summarised in (Table 2). In most of the media, the colonies grew into compact c a l l i and produced masses of green nodules. The nodules f a i l e d to regenerate into shoots a f t e r three subcultures on fresh aliquots of the same medium from which they had developed. The modified Murashige and Skoog (1962) agar medium containing BAP 1.13 mg/L and NAA 0.02 mg/L was the best f o r shoot regeneration from root and cotyledon protoplast-derived tissues of Linum. I t was suitable for shoot regeneration in s t e m ~ n t s of Brassica napus (Kartha et a l , 1974) and suitable f o r s-TT6-6-lTregeneration in stem and callus explants of Linum usitatissimum (Murray et a l , 1977). Root ~tion was induced when shoot tips with 3-4 leaves were transferred to MS based agar, without phytohormones, and h a l f strength of B5 based agar with 0.2 mg/L IAA (Figs. l - 5 ) . Previously i t has been emphasised that the use of protoplasts isolated from seedling roots (Xu et a l . 1981, 1982) and from seedling cotyledons (Lu et a l . 1982) could be p a r t i c u l a r l y advantageous when somatic hybridization assessments are being undertaken. The use of germinating seeds overcomes the d i f f i c u l t i e s often encountered with the production of l e a f material, and enables an adequate supply of experimental material to be produced rapidly with the minimum of resources. I t has also been suggested for somatic hybridization assessments that protoplasts from roots and e t i o l a t e d cotyledons could be used, instead of cell suspension protoplasts, f o r fusion with green cotyledon l e a f protoplasts or l e a f mesophyll protoplasts (Lu et a l , 1982). Heterokaryons can be i d e n t i f i e d and isolated manually (Patnaik et a l , 1982). Moreover, the y i e l d of cotyledon protoplasts is also often adequate for genetic transformation studies (Cocking et a l , 1981). These present studies on plant regeneration from root and cotyledon protoplasts of Linum usitatissimum w i l l provide an adequate cultural for such genetic manipulation assessments. The culture c a p a b i l i t i e s of the other species of Linum are also currently being investigated.
Fig. I . Freshly isolated protoplasts. a-Root~ XI50o b-Hypocotyl~XlO0. C-Cotyledon,X125. Fig. 2. Cell colonies derived from hypocotyl protoplasts a f t e r 7 days of culture on l i q u i d medium. X300. Fig. 3. Cell colonies derived from cotyledon protoplasts a f t e r 15 days of culture on solid medium: 5 cm diameter dish. a-culture in KM8P:KM8 medium, b-culture in MB5P medium. Fig. 4. P r o l i f i c shoot regeneration from callus derived from cotyledon protoplasts.
Table 2. Response of d i f f e r e n t explant protoplast-derived callus to various media. Medium
Protopl ast-derived callus
Composition
Root MSP-I MSP-3 MSD-3 MSD-4 MSF MSZ B5-1 B5-2
Hypocotyl Cotyledon
Murashige and Skoog 1962 (2.0 mg/L NAA and 0.5 mg/L BAP) Murashige and Skoog 1962 (0.I mg/L NAA and 0.5 mg/L BAP)
A,R
A,R
A,R
+,S
Murashige and Skoog 1962 (2.0 mg/L IAA and 1.0 mg/L BAP) Murashige and Skoog 1962 (0.05 mg/L N/~A and 0.5 mg/L BAP)
A,R,+
+,R A,R+
+,R A,R,+
A,R,+
A,R,+
A,R,+
++,S
+,R
++
Murashige and Skoog 1962 (0.02 mg/L NR~Aand 1.13 mg/L BAP) Murashige and Skoog 1962 ( I . 0 mg/L Zeatin) Gamborg et a l , 1968 (0.02 mg/L NAA and 2.0 mg/L BAP) Gamborg et a l , 1968 (0.I mg/L NAA and 0.5 mg/L BAP)
R= Roots S= Shoots A= Abundant callus ++ = Large green nodular callus.
+ = small
,S
++
++
++
+
+
+
+ +
+
green nodular callus
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