Welsh Plant Breeding Station, Aberystwyth, Dyfed, Wales, UK. 1. INTRODUCTION. A prerequisite for the use of many genetic manipulation techniques is.
Plant Cell, Tissue and Organ Culture 12:13%140 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands
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PLANT REGENERATION FROMCELL SUSPENSION PROTOPLASTS OF FESTUCA ARUNDINACEA SCHREB., LOLIUM PERENNE L. AND L.MULTIFLORUIQ[-L-X]~7-. S. J. Dalton AFRC I n s t i t u t e for Grassland and Animal Production, Welsh Plant Breeding Station, Aberystwyth, Dyfed, Wales, UK
1.
INTRODUCTION A prerequisite for the use of many genetic manipulation techniques is the a b i l i t y to regenerate plants from protoplasts, but in the cereals and grasses, plants have been regenerated in this way in only six species ( I ) . Of these, only Oryza (2,3,4,5,6) and Saccharum (7) have been successfully transferred ~ i 1 . In v i t r o p l a ~ r e e forage grasses: Festuca arundinacea ( t a l l fesc~),--T~lium perenne (perennial r y e g r a s s ) ~ m u l t i f l o r u m ( I t a l i a n ryegrass),a-~ffav-e-n~regenerated from protoplasm-derived cell colonies and green Festuca and L.multiflorum plants established in soil (8). 2. CELLSUSPENSION AND PROTOPLAST CULTURE 2.1. Cell suspension i n i t i a t i o n and culture The regenerating protoplasts were isolated from young embryogenic cell suspensions i n i t i a t e d directly from groups of chopped mature embryos. The three species used were the most responsive of seven Lolium, Festuca, Dactylis and Phleum grass species tested with this m e t h o - - d - ' o l r - s u ~ n In~-6Tt-Ca-t-Ton. ~ r i g i n a l reasons for establishing cell suspensions of outbreeding species from a number of mature embryos were a) to use a convenient source of meristematic tissue, b) to save time by eliminating the callus phase and c) to obtain a ' t y p i c a l ' suspension of each species by culturing a mixture of genotypes. However, ~ t h i n each species, the cell suspension lines were as ~rphologically variable as those begun from c a l l i of single genotypes. Iso-enzymeanalysis of some of the plants regenerated from chopped embryo derived cell suspensions indicated that heterogeneity within individual cell suspensions (or at least of the regenerating tissues) was low. Cell suspensions were i n i t i a t e d by culturing up to 30 mature ~nbryos (chopped up with a scalpel) i n liquid medium containing a high concentration of 2,4-D (10 mg I - I ) . Fresh culture medium containing the normal cell suspension concentration of 2,4-D was added after 10 days (8). Once established the cell suspensions were subcultured weekly by transferring sma11, dense colonies to 80% fresh culture medium. 2.2. Protoplast isolation and culture Protoplasts were isolated four days after subculture and could be isolated as soon as four weeks after suspension i n i t i a t i o n . However, the yields were generally low (less than 3 x 106/g) until about 20 weeks after suspension i n i t i a t i o n . A higher proportion of the protoplasts isolated from the young cell suspensions were small and dense compared with those isolated from older, friable suspensions. Protoplasts i n i t i a l l y isolated from young (13-20 week old) Festuca and L.perenne suspensions did not survive in liquid culture m e d i a ~ m o s t sure for callus colony formation from protoplasts isolated from ol der
138 cell suspensions containing 3% sucrose and 10% mannitol (835-870 mOsm). However, colonies were formed when the protoplasts were cultured in a similar medium (PC4) of higher osmotic potential (895 mOsm), achieved by the addition of 6.84% glucose and 5% mannitol (8). Protoplasts of the three species isolated from most of the cell suspension lines tested were capable of producing cell colonies when cultured in medium PC4. Inconsistent results were obtained when protoplasts were cultured in medium s o l i d i f i e d with seaplaque agarose. 2.3. Protoplast plating efficiency and colony culture Festuca protoplasts were the most responsive of the three species tes~th protoplasts isolated from 15 out of 20 cell suspension lines achieving plating efficiencies of 0.1-1.0% (400-4000 colonies per plate) (Table I ) . Protoplasts from only one out of nine L.perenne suspension lines and two out of six L.multiflorum suspension lines were capable of the same efficiency. Strands of single cells, friable and compact cell colonies were formed. However, less than 50 colonies per plate were compact colonies, depending on the cell suspension line and i t s age. TABLEI. Plating efficiency of cell suspensionprotoplasts
Species F.arundinacea (20) C.pe~nne (9) U.~----l~r~Forum(6)
No. of protoplast isolations per cell suspension line (total) 1-15 (78) 1-9 (22) 2-4 (17)
No. of protoplasts(xlO6) cultured per cell suspensionline (total) 0.1-91 (460) 2-96 (230) 8-31.5 (107)
NO. of lines with 0.1-1.0% plating efficiency 15/20 I/9 2/6
The protoplast-derived colonies were plated onto s o l i d i f i e d medium of the same composition as medium PC4 but without mannitol (8). After several weeks culture, any embryogenic and nodular c a l l i developing from the compact colonies were transferred to regeneration medium. Green and white shoots emerged f r o m embryolds in Festuca and L.perenne, but embryogenesis was less evident in L.multiflorum. 3. 3.1
REGENERATIONFROMCELL SUSPENSION AND PROTOPLAST-DERIVED COLONIES Comparisonbetween cell suspension and protoplast-derived colonies Plated protoplast-derived cell colonies were generally capable of the same merphogenesis as colonies derived f r o m the respective cell suspension lines. Most cell suspensions were i n i t i a l l y able to regenerate green shoots when plated, but the regenerative a b i l i t y declined with age (Table 2). The number of shoots produced declined and the proportion of shoots which were albino and the proportion of friable tissue increased. However, for a majority of the cell suspension lines which produced responsive protoplast-derived colonies, shoots could be obtained from isolations performed after the suspensions had apparently stopped regenerating. These shoots were usually albino. Protoplastderived colonies regenerating green shoots tended to be formed in isolations performed within the regenerative l i f e of the cell suspension as determined by plating. 3.2. Plant regeneration from F.arundinacea cultures Protoplasts were isolated from 20 cell suspension lines 6-122 weeks from suspension i n i t i a t i o n . Suspension colonies of 12 of the lines were able to regenerate green shoots when plated, but this response ceased
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TABLE 2.
The effect of cell suspension age on shoot formation from plated cell suspension and protoplast-derived colonies Age range in weeks of cell suspension at the time: Protoplasts were isolated Shoot regeneration from which subsequently developed plated cell suspension shoot forming cell colonies colonies ceased Green White Green White
F.arundinacea (20) ~.perenne (9} ~.mu-~T~Tfl-orum (6)
18-34 (6) 24-30 ( I ) 25-33 (1)
11-63+(12) 21-63+ (5) 21-33 (I)
12-36 (12) 11-48 (11) 15-29 (6) 19-63 (7) 12-43 (5) 18-43 (3)
() Number of cell suspension lines a f t e r a maximum of 36 weeks (Table 2). Eleven lines produced albino shoots and this response generally continued for a longer period, ceasing after a maxium of 48 weeks. Two lines produced only embryogenic tissue and this response ceased after 14 and 15 weeks. However, both of these lines produced shoots from protoplast-derived colonies (Table 3). A green shoot was also produced from protoplast-derived colonies of another suspension which was apparently capable of albino shoot regeneration only. Altogether, shoots regenerated f r o m protoplast derived cell colonies of 12 cell suspension lines. N i n e of these responsive lines regenerated shoots from protoplast isolations performed after the cell suspension had apparantly ceased regenerating shoots. The great majority of shoot forming colonies were derived from only two lines (261 green and 510 white). O v e r 120 plants were established in soil, but require vernalisation before the a b i l i t y to set seed can be determined. 3.3. Plant regeneration from L.perenne cultures Protoplasts were isolated from nine cell suspension lines 11-63 weeks from suspension i n i t i a t i o n . Suspension colonies of six of the lines regenerated green shoots when plated, but this response ceased after a maximum of 29 weeks, while seven lines which regenerated white shoots when plated, continued this response until a maximumof 63 weeks (Table 2). One line which did not regenerate shoots and did not appear to be embryogenic, nevertheless produced three protoplast-derived colonies with albino shoots. In the most responsive line, white shoots formed from 371 protoplast-derived colonies obtained in isolations performed 22-63 weeks after suspension i n i t i a t i o n (Table 3). This was also the only line to have a plating efficiency above 0.1%. Two green plantlets formed from colonies obtained from protoplasts isolated 24-30 weeks after suspension i n i t i a t i o n did not survive in soil. Three more responding lines produced 15 colonies with white shoots from protoplasts isolated 21-34 weeks after suspension i n i t i a t i o n ; in two of these this was after the respective cell suspensions had ceased regenerating shoots. Some protoplast-derived colonles had green areas but these did not develop further. 3.4. Plant regeneration from L.multiflorum cultures Protoplasts were isolated from six cell suspension lines 12-33 weeks after suspension i n i t i a t i o n . Suspension colonies of five of the lines retained the capacity for green shoot regeneration for up to 43 weeks while three of the lines regenerated white shoots for up to 43 weeks (Table 2). One suspension produced only embryogenic tissue up to 18 weeks. Protoplasts of five suspensions formed cell colonies. However,
140 TABLE 3.
Species F.arundlnacea (20) L.perenne (9) L.multiflorum (6)
Shoot regeneration from c e l l suspension protoplast-derived colonies No. of protoplast derived colonies with shoots Green White 159" (1) 122 (5)
379* (1) 118 (11)
2* (1) 0
371" (1) 18 (4)
24* (1)
4
(1)
No. of green plants established in soil 77* (1) 47 (3) 0 0 5* (1)
() Number of cell suspension l i n e s ; * Most responsive l i n e . colony formation was generally low (less than 0.1%) and only the two lines with the highest plating efficiencies produced protoplast derived colonies capable of shoot formation (Table 3). Colonies from one line formed albino shoots although these were from isolations performed after the cell suspension had ceased regenerating. Colonies of the other line produced green shoots and five green plants were established in soil. 4. DISCUSSION Further work is required to improve the rate of plant regeneration and the applicability of this technique to a wider range of genotypes. However, these results indicate that green plants can be regenerated from gramlneous protoplasts if they are isolated from fast growing, young, embryogenic cell suspensions, before the ability of cell suspension colonies to regenerate green plants has ceased (typically up to 30 weeks after suspension initiation). The embryogenic protoplasts isolated from such cell suspensions respond when cultured in high osmotic potential medium achieved by the addition of glucose. Green and albino shoots have been obtained from protoplast-derlved colonies of all three species although only plants of Festuca and L.multiflorum have been established in soil. L.multiflorum cu-~ll~-s may-be inherently less prone to produce albino shoo-ts than L.perenne although the L.multlflorum material used was selected for embr~ogen-e'nT~response whil~ the Festuca and L.perenne material were ordinary cultlvars (WPBS cultlvars--~--~-.170 a n ~ u - A - d ~ respectively). The tendency for gramineous cultures and L.perenne in particular, to rapidly become albino may now be one of the ~ ~ e m s slowing further progress in this family. 5. I. 2. 3. 4. 5. 6. 7. 8.
REFERENCES Vasil IK: J. Plant Physiol. 128, 193-218, 1987. Abdullah R, EC Cocking, JA Thompson: Blotechnology 4, 1087-1090, 1986. Coulibaly MY, Y Demarly: Z. PflanzenzBchtg. 96, 79-81, 1986. Kyozuka K, Y Hayashl, K Shlmamoto: Hol. Gen. Genet. 206, 408-413, 1987. Toriyama K, K Hlnata, T Sasaki: Theor. appl. Genet. 73, 16-19, 1986. Yamada Y, Y-Q Yang, D-T Tang: Plant Cell Reports 5, 85-88, 1986. Srlnivasan C, IK Vasil: J. Plant Physiol. 126, 41-48, 1986. Dalton, SJ: J. Plant Physiol. (in press).
