Isolation, culture and regeneration of protoplasts from potato and several related Solanum species

Plant Science, 39 (1985) 67--74 Elsevier Scientific Publishers Ireland Ltd.

67

ISOLATION, CULTURE AND REGENERATION OF PROTOPLASTS FROM POTATO AND SEVERAL RELATED S O L A N U M SPECIES*

G.T. HABERLACH a'c, B.A. COHEN c'***, N.A. REICHERT b,t, M.A. BAER c, L.E. TOWILL a , b ' t t and JOHN P. HELGESON a,c,**

aUnited States Department o f Agriculture, Agricultural Research Service, and b Departments o f Horticulture and cplant Pathology, University o f Wisconsin, Madison, WI 53706 (U.S.A.) (Received December 5th, 1984) (Revision received February 4th, 1985) (Accepted February 8th, 1985) Thirty*six cultivars, breeding lines and accessions from five Solanum species were examined for isolation, culture and regeneration of leaf mesophyll protoplasts. These included diploid and tetraploid Solanum tuberosum clones as well as accessions of S. brevidens, S. demissum, S. etuberosum and S. pennellii. Test lines were chosen because of desirable disease resistances or markers that could be used for studies of somaclonal variation and somatic fusions. Stock plants and test lines were grown aseptically on a modified Murashige and Skoog (MS) medium. These plants were particularly good sources of protoplasts and routinely yielded 10--20 million protoplasts from 1 g of leaf tissue. All tested clones yielded some viable protoplasts and all but three yielded protoplasts that were capable of at least one cell division. Callus tissues were obtained from 32 of the 36 clones and 22 of these produced shoots.

Key words: Solanum: potato; protoplast isolation; protoplast regeneration

Introduction

The tuber-bearing Solanum species and their close relatives are rich sources of germ*Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. **To whom reprint requests should be sent. ***Present address: Agracetus Madison Corporation, 8520 University Green, Middleton, WI 53562, U.S.A. tPresent address: Advanced Research Lab., Agrigenetics Corporation, 5649 E. Buckeye Road, Madison, WI 53716, U.S.A. t t p r e s e n t address: USDA, ARS, Dept. of Horticulture, Colorado State Univ., Fort Collins, CO, 80523, U.S.A. Abbreviations: ABA, absiscic acid. BA, benzylaminopurine; CL, cell layering; Con, conditioning; Cul, culture; Dif, differentiation; GA3, gibberellic acid; IAA, indoleacetic acid; MBC, meristem bud culture; MS, Murashige and Skoog [12 ]; NAA, naphthaleneacetic acid; Prop, propagation; Res, reservoir; Rin, rinse.

plasm for potato improvement. However, sexual incompatibility barriers may interfere with incorporation of new traits (e.g. stress or disease resistance) into potato cultivars. Somatic hybridization [1] might provide alternative methods for utilizing this germplasm. A prerequisite for such somatic manipulations is the successful isolation and culture of protoplasts and the redifferentiation of the cultured cells into new plants. For this reason, there has been considerable interest in the regeneration of plants from potato protoplasts [2--12]. We have been examining selected lines and species from the large collection maintained by the Inter-Regional Potato Introduction Project (IR-1) at Sturgeon Bay, WI. The selections include wild Solanum species, cultivars, cultivar haploids and other materials (Table I). Our goal is to obtain a collection of genetically diverse lines that are facile producers of protoplasts which will readily regenerate.

0168-9452/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

68 Table I.

Materials used in tests.

Species

Identifiera

C h r o m o s o m e no.

A g r o n o m i c characters

S. brevidens

PI 2 1 8 2 2 8 PI 2 4 5 7 6 3

24 24

Resistant to p o t a t o leaf roll virus and frost

S. demissum

PI 205625

72

Resistant to

Phy toph thora infestans S. etuberosum

PI 2 4 5 9 2 4 PI 245939

24 24

Resistant to frost Resistant to frost

S. pennellii

PI 2 4 6 5 0 2

24

R e s i s t a n t to tentoxin from

Alternaria alternata S. tuberosum

Russet Burbank Katahdin

48 48

Common commercial cultivars

PI PI PI PI

203900 423654 303146 215618

48 48 48 48

Late blight differentials b

For For For For

PI PI PI PI

319426 319428 256977 319433

48 48 48 48

Mexican cultivars field resistance to P. infestans

' Bertita' 'Dorita' 'Elenita' 'Greta'

Adg. 156 hap. US-W 13,114 US-W 13,067

24 24 24

Haploids

F r o m Gp. A n d i g e n a F r o m cv. S u p e r i o r F r o m cv. K e n n e b e c

US-W US-W US-W US-W

5328.4 9545.75 9545.99 9545.46

24 24 24 24

Haploid-hybrids

(Gp. Phureja x Gp. Tuberosum haploid), red t u b e r flesh

US-W US-W US-W US-W

9310.3 9587.24 9587.31 9587.33

24 24 24 24

Haploid-hybrids

{Gp. Phureja x Gp. Tuberosum haploid), yellow t u b e r flesh lines

77d9 77-5 75-15 75-21

24 24 24 24

M e t r i b u z i n test lines, Gp. Phureja/Stonotomum diploids f r o m H. De J o n g

MeMe Meme Meme Meme

77-1 77-16

24 24

Metribuzin-sensitive lines, Gp. Phureja

meme meme

F 51013 F 65089 Voran

48 48 48

M e t r i b u z i n sensitive M e t r i b u z i n sensitive M e t r i b u z i n sensitive

Line Line Cultivar

race race race race

R, R4 R~ R,,.,,~, 4

a All PI ( p l a n t i d e n t i f i c a t i o n n u m b e r ) and US-W labeled materials o b t a i n e d f r o m Inter-Regional P o t a t o I n t r o d u c t i o n S t a t i o n , S t u r g e o n Bay, WI; all m e t r i b u z i n differential materials o b t a i n e d f r o m H. De Jong, Agriculture Canada~ F r e d e r i c t o n , N e w Brunswick. b Lines s u s c e p t i b l e to particular race o f P. infestans indicated.

69

Materials and methods

These materials will then be used in studies of somatic fusions [13, see also accompanying paper 14]. We now report the results of an evaluation of 36 selections from the IR-1 collection of Solanum species and S. tuberosum cultivars. Preliminary reports have been given on the development of the methods and on initial results with various cultivars, lines and species [15,16]. Table II.

Media for preparation

NH4NO ~ KNO3 CaC12 • 2 H ~ O KH2PO, H3BO ~ KI Na2MoO4 • 2H20 CoC12 • 6 H 2 0 MgSO4 • 7H:O MnSO 4 • 4H~O ZnSO, • 7H20 CuSO, • 5H~O FeSO 4 • 7H20 NaeEDTA Sucrose NH4CI Adenine sulfate Thiamine • HCI P y r i d o x i n e • HC1 Nicotinic acid Biotin Glycine Folic acid Casein hydrolysate Agar IAA t-Zeatin BA Myo-inositol

The methods for isolation, culture and regeneration were adapted for use with asceptically grown plants from the procedures of Shepard and Totten [8,10]. Media The media used for isolating protoplasts and for regenerating plants are listed in

and culture of protoplasts

from leaves of Solanum species.

MS salts and vitamins mg/i

Con

mg/1

Enzyme digest .mix mg/l

1650 1900 440 170 6.2 0.83 0.25 0.025 370.0 22.3 8.6 0.025 27.86 37.26 30 g/l . . . . 0.1 0.5 0.5 -2.0 --1 0 g/1 . . . . -100

. . 190 44 17 0.62 0.083 0.025 0.0025 37 2.23 0.86 0.0025 2.786 3.726 -. . 0.05 0.05 0.5 0.005 0.2 0.05 100 -. . 0.5 10

. . . . . 190 3800 7600 1900 44 880 1760 440 17 340 680 170 0.62 1.55 3.10 3.10 0.083 0.21 0.42 0.42 0.025 0.063 0.13 0.13 0.0025 0.0063 0.013 0.013 37 741 1481 370 2.23 5.58 11.16 11.16 0.86 2.15 4.30 4.30 0.0025 0.0063 0.013 0.013 2.786 6.97 13.94 13.94 3.726 9.32 18.64 18.64 103 g/l 103 g/l 6 8 . 6 6 g/1 1 7 . 1 7 g/1 . . . . . . ---0.5 ---0.5 ---5 ---0.05 ---2 ---0.5 ---100 --7 g/l 10 g/l . . . . . . . . 0.5 0.5 0.4 0.4 --4.505 g/l-

2.0

Rin

CL

Res

Cul

Dif

mg/1

rag/1

mg/l

mg/l

mg/1

1900 440 170 3.10 0.42 0.13 0.013 370 11.16 4.30 0.013 13.94 18.64 2 . 5 g/1 107 40 0.5 0.5 5 0.05 2 0.5 100 1 0 g/1

1900 440 170 3.10 0.42 0.13 0.013 370 11.16 4.30 0.013 13.94 18.64 2.5 g/l 267.5 80 0.5 0.5 5 0.05 2 0.5 100 10 g/l 0.1 2.5 -100

NAA

--

PVP

--

--

20 g/l

.

Cellulysin

--

--

5 g]l

.

Macerase D-mannitol Sorbitol Xylitol

-. . .

-. . .

1 g/l

.

. . .

2.0

. . .

2.0

1 . .

.

0.5 100

1

0.i

.

.

.

.

.

.

. . . 4 . 5 5 g/1 45.55 g]l 4 . 5 5 g/1 - 3.8 g/l --

--

54.7 g/l ---

36.4 g/l ---

70 Table II. The meristem bud culture (MBC4) medium was MS medium [17] with 2.9 pM indoleacetic acid (IAA), 2.3 pM zeatin and 0.52 uM gibberellic acid (GA3). The propogation (Prop) medium was MS medium with 2X the level of CaC12 and KH:PO4, 2.7 t~M thiamine--HC1, 0.5 uM pyridoxine HC1, 0.81 #M nicotinic acid and 5.3 pM glycine. Agar (Difco-Bacto-Agar5) concentrations were 0.6 and 0.7% (w/v) respectively, for MBC and Prop medium. The medium for floatation of leaves contained CaC12 and NH4NO3 (1 mM each) as well as 10.7 uM napthaleneacetic acid (NAA) and 4.4 t~M benzylamino purine (BA) [10]. All media were adjusted to pH 5.8 + 0.2 with 1 N NaOH or KOH.

Stock plants derived from seeds Seeds were surface sterilized for 30 min with 0.5% sodium hypochlorite ( 1 : 1 0 dilution of household bleach), rinsed with sterile, distilled water and incubated overnight at room temperature (22°C) in a sterile solution of 5.7 pM GA3. The seeds were rinsed with sterile, distilled water and placed on 6 ml of MBC medium in 50 X 15 mm plastic plates (Falcon No. 1007). They were allowed to germinate at 24°C under cool-white fluorescent light (about 75 t~E m -2 s-~). Seedling shoot tips were excised and rooted in test tubes containing MS medium [17] with 4% (w/v) sucrose. Stock plants from tubers Tubers were planted in soil mix ( 1 : 1 : 1 , peat/sand/field soil) in pots in a greenhouse. Axial buds from these plants were excised aseptically and cultured on MBC medium. Shoot tips about 1 cm long were then excised from the developing plants and cultured in test tubes on MS medium with 4% sucrose. Clonal propagation o f stock lines Sterile stock plants from test tubes were divided into 1--2 cm segments with a nodal bud and leaf on each section. These nodal

sections were planted upright in 20 X 150 mm test tubes containing 10 ml of Prop medium and the tubes were topped with clear plastic closures (Kimkap No. 20, Kimble). These plants were grown at 24°C under cool-white fluorescent light (about 114 # E m -2 s -~, 16-h photoperiod) and were subcultured every 4--6 weeks. For protoplast isolations, 9 such nodal sections were planted in a 9.5 X 9.5 X 10 cm 'PlantCon' containers (Flow Laboratories Inc.) containing 100 ml of Prop medium. The 'PlantCons' were sealed with Parafilm strips and plants were grown under the same conditions as stock clonal lines for 4--6 weeks.

Protoplast isolation Leaves (1--3 g) were excised from plants, weighed in sterile, tared petri dishes and placed, topside down, on 3 layers of filter paper in 20 X 150 mm glass petri plates containing 30 ml of floatation medium and sealed with Parafilm strips. After 48 h in the dark (20--22°C) the leaves were placed in a 250-ml Erlenmeyer flask containing 125--150 ml of conditioning medium (Table II) and incubated for 24 h at 4°C. The leaves were then chopped with a straight edge blade (Aluminium Pathco knife) and placed in 100 ml of digest medium (Table II} in a 1000-ml filter flask. Leaves were vacuum infiltrated with the digest medium and were then incubated for 16 h (28°C, 60 pE m -2 s -~ cool-white fluorescent light) while gently swirling in a water bath shaker (New Brunswick model G-76) set at 40 rev./min. At the end of the incubation period, flasks were swirled for 40 s at 200 rev./min on a shaker (New Brunswick G-33). The digested leaves were filtered through a 94-pm mesh stainless steel screen (Bellco Glass Inc. No. 1985--00150) and the filtrate was transferred to sterile Babcock bottles (Kimble No. 1000). The bottles were topped with sterile glass dram vials and centrifuged at 350 X g (1300 rev./min International model HN centrifuge). The protoplasts were rinsed by resuspension of the cells in rinse (Rin) medium and recentrifugation (1--2 times). After determining the number of protoplasts

71 by a h e m o c y t o m e t e r count, the volume was adjusted with Rin medium to give a protoplast concentration of 106 m1-1 and the protoplasts were allowed to stand 1--2.5 h at room temperature. Viability determinations were made by fluorescein diacetate staining [18].

Culture and differentiation o f protoplasts The protoplast preparation was diluted with cell layering (CL) medium to 2.4 × l 0 S cells ml -~ and then with two parts of warm (45--50°C) CL medium containing 1.05% agar. Three milliliters of this suspension was spread over 10 ml of solidified reservoir (Res) medium in a 100 × 15 mm plastic petri plate (Falcon No. 1029). At least 6 and usually 10 plates were prepared for each test line. The bilayer plates were then sealed with Parafilm and incubated for 10--14 days at 24°C under cool-white fluorescent light (light from bulbs was reduced to 20 ~E m -2 s -1 by cheese cloth). When the protoplasts had formed cell walls and many cells had also divided (2--3 weeks depending on test line), the bilayer plate was divided into 6 equal wedges and each wedge was layered onto 20 ml of agar-solidified culture (Cul) medium in a 100 X 15 m m petri plate. The plates were then incubated at 24°C under cool-white fluorescent light (about 100 pE m -: s -1, 16-h photoperiod) for 2 weeks. The microcalli which formed were transferred (100 calli/plate) to fresh Cul medium and incubated under the above conditions for another 2 weeks. The bright green calli that developed at this stage were transferred to differentiation (Dif) medium (about 50 calli/plate) and incubated as with Cul. Plates were observed periodically for the appearance of shoots. Shoots were excised from the callus and rooted in test tubes with Prop medium. Results Thirty-six Solanum lines from five species were tested. Each line was tested at least twice and representative data from 34 of these lines are given in Table III. Two addi-

tional S. tuberosum lines, cv. Russet Burbank and PI 203900, were used extensively in testing the m e t h o d [15] and were generally included as controls in experiments. These two lines were compared directly in six experiments. Russet Burbank and PI 203900 produced 10.5 + 1.7 (S.E.) and 10.6 + 3.2 million protoplasts/g of leaf, respectively. An average of 23 +- 7% of the Russet Burbank calli and 47 +-12% of the PI 203900 caUi yielded shoots after plating on Dif medium. Viable protoplasts were obtained from clones of each of the 36 tested accessions from five species. Three lines, the Mexican cv. Elenita (PI 256977), a haploid from .cv. Kennebec, and 77-16, a diploid from the Canadian collection of H. De Jong, failed to give protoplasts that were capable of undergoing cell division. US-W 9587.24, another haploid from the IR-1 collection, gave protoplasts that were capable of a few cell divisions but did not produce macroscopic calli. The remaining 32 lines gave at least a few macroscopic calli per plate. Of the 32 lines that formed calli, 10 failed to give shoots after transfer to Dif medium. With three of these, PI 319426, US-W 9587.33 and 75-15, the numbers of calli produced were so low that only a few could be tested; none of these produced shoots. However, in the other seven cases, several hundred calli were tested from each line. Of these lines, four iS. demissum, cv. Superior haploid, 77-1, and cv. Voran) produced calli t h a t died 4--6 weeks after transfer to Dif medium. The other three lines (PI 303146, 77-5, cv. Greta) yielded calli that grew for several m o n t h s but produced no shoots. A total of 22 of the 36 test lines gave shoots and the percent of the calli t h a t formed shoots varied widely between various lines (Table III). For example, one S. brevidens accession, PI 245763, yielded shoots on 41% of the calli whereas only 1% of the calli from another accession of the same species, PI 218228, formed shoots. Particularly high yields of shoots were obtained with two S. etuberosum accessions (PI 245924 and PI

72 Table III. E v a l u a t i o n o f S o l a n u m species for p r o t o p l a s t yield a n d survival, division of surviving cells and regenera t i o n o f calli derived f r o m p r o t o p l a s t s .

Test lines

Protoplast yield no. × 10-S/g

S. brevidens PI 2 1 8 2 2 8 240 PI 2 4 5 7 6 3 266 S. d e m i s s u m PI 2 0 5 6 2 5 30 S. e t u b e r o s u m PI 2 4 5 9 2 4 50 PI 2 4 5 9 3 9 67 S. p e n n e l l i i PI 2 4 6 5 0 2 92 S. t u b e r o s u m Katahdin 96 PI 4 2 3 6 5 4 208 PI 3 0 3 1 4 6 105 PI 2 1 5 6 1 8 88 PI 3 1 9 4 2 6 19 PI 3 1 9 4 2 8 45 PI 2 5 6 9 7 7 26 PI 3 1 9 4 3 3 27 A n d i g e n a hap. 156 102 S u p e r i o r hap. 7 3 4 . 9 110 K e n n e b e c hap. 7 3 1 . 3 172 US-W 5 3 2 8 . 4 82 US-W 9 5 4 5 . 7 5 110 US-W 9 5 4 5 . 9 9 97 US-W 9 5 4 6 . 4 6 3 US-W 9 3 1 0 . 3 149 US-W9587.24 14 US-W 9 5 8 7 . 3 1 8 US-W 9 5 8 7 . 3 3 27 77-19 107 77-5 83 75-15 8 75-21 19 77-1 199 77-16 60 F 51013 49 F 65089 50 Voran 20182 60

Survival %a

Division %b

Calli/plate no.C

Shoots %d

18 30

3 15

200 >4000

1 41

20

9

3000

0

65 80

52 64

>4000 >4000

36 47

21

1

602

9

53 52 55 70 1 45 25 30 65 46 2 60 20 90 5O 2O 50 19 20 55 10 6 80 80 1 22 70 20

21 65 33 1 1 23 0 1 33 9 0 24 12 68 45 6 5 2 1 22 1 1 28 72 0 3 28 18

3000 2000 600 185" 2* 2166 0* 58 2218 1299 0* 45* 526 3900 3600 >4000 0* 63 2 162 142" 4 4 >4000 0* 141" >,t000 3000

5 81 0 17 0 2 0 0 44 0 0 69 5 5 51 9 0 3 0 2 0 0 22 0 0 13 16 0

a P e r c e n t a g e o f p r o t o p l a s t s s h o w i n g viability as j u d g e d b y m i c r o s c o p i c e x a m i n a t i o n 10 or m o r e days a f t e r isolat i o n a n d plating. b P e r c e n t a g e o f p r o t o p l a s t s s h o w i n g a t least o n e cell division a f t e r plating. c N u m b e r s of calli c o u n t e d o n C L / R e s plates ( n u m b e r s greater t h a n 4 0 0 0 were difficult to c o u n t accurately). E x c e p t w h e r e i n d i c a t e d b y a n asterisk (*) 2 4 0 0 0 0 p r o t o p l a s t s were initially p l a t e d o n each plate. d P e r c e n t a g e o f t h o s e calli t h a t f o r m e d s h o o t s w i t h i n 1 5 - - 2 5 weeks a f t e r t r a n s f e r t o d i f f e r e n t i a t i o n m e d i u m . * E x p e r i m e n t w i t h 2 4 0 0 0 0 p r o t o p l a s t s p l a t e d per p l a t e failed t o p r o d u c e s h o o t s . Data here are for an e x p e r i m e n t with plating of 480 000/plate.

73 245939), an R-4 late blight differential line of S. tuberosum (PI 423654) and several haploid lines (Andigena 156, USW 5328.4 and USW 9545.46). Discussion Although the procedures used for isolation of protoplasts generally are those of Shepard and his co-workers [8--11], several differences should be noted. Plants for protoplast isolations were growh in test tubes, 'PlantCons' (Flow lab) or 'GA-7' containers (Magenta). This proved to be a very useful way of growing and maintaining m a n y clones in a relatively small area under precisely controlled environmental conditions. Two 'PlantCons' occupying a total shelf area of only 100 cm 2 each can provide ample leaf material for an experiment. In addition, the yields of protoplasts from the axenically-grown plants was very high. For exar{aple, both clones of S. brevidens produced over 20 million protoplasts per gram of leaf tissue (Table III). This is more than 10-fold the m a x i m u m yield of 1--1.5 million per gram tissue reported by Barsby and Shepard [ 3 ] with this species. The results of our tests indicate that this m e t h o d is suitable for a wide range of potato lines and cultivars as well as other Solanum species. Overnight (about 16 h) rather than 3--5 h incubation with digestion enzymes was routinely used. This allowed harvest of fresh protoplasts at the beginning of each working day. Under these conditions, the inclusion of cytokinin and auxin in both rinse and digest media consistently resulted in higher protoplast yields. This m a y be due to prevention of p h y t o h o r m o n e depletion during incubation. The shorter incubation periods, about 4--6 h, used by Shepard and his co-workers might n o t result in such deficiencies. In addition to the procedural differences noted above, some of the media were modified from those initially used by Shepard and Totten [8] and Shephard [10]. Results from tests of the media formulations [15,16] prompted us to increase the concentration of

zeatin in Dif medium. Also, because of its inhibitory effect on colony growth and regeneration, we omitted Res. Since our initial report [15], Shepard [11] has reported that 9.1 uM zeatin (2 rag/l) is effective for several potato cultivars. Abscisic acid (ABA) was also omitted in our optimal formulation because it had no beneficial effect when the zeatin concentration was optimal [15]. The 36 different lines examined in our study include representatives of S. tuberosum Gp. tuberosum cultivars, late blight differential lines, other Solanum species and Gp. andigena and Gp. tuberosum haploids derived from S. tuberosum tetraploids. In all cases, we obtained viable protoplasts and, in all but four cases, sufficient cell division ensued to yield a macroscopic callus. This latter point is particularly important as even calli that fail to regenerate may be very useful. For example, De Jong's 77-1, one of the lines that did not regenerate under our standard conditions, formed large numbers of healthy calli. This line has been used in a somatic fusion experiment with S. brevidens, PI No. 245763. Progeny with the characteristics of both fusion partners have been obtained [14]. Acknowledgements We t h a n k H. De Jong and R.E. Hanneman, Jr. for our Solanum materials. References 1 0 . Schieder and I.K. Vasil, in: I.K. Vasil (Ed.), Perspectives in plant cell tissue culture, Int. Rev. Cytol., Suppl. 11B, Academic Press, N e w York, 1980, p. 21. 2 S. Austin and A.C. Cassells, Proc. R. Ir. Acad., (1983) 53. 3 T. Barsby and J.F. Shepaxd, Plant Sci. Lett., 31

(1983) 101.

4 H. Binding, R. Nehls, O. Sehieder, S.K. Sopory and G. Wenzel, Physion. Plant., 43 (1978) 52. 5 R.G. Butenko, A.A. Kuchko, V.A. Vitenko and V.A. Aventisov, Soy. Plant Physiol., 24 (1977) 660. 6 R.E. Gunn and J.F. Shepard, Plant Sci. Lett., 22 (1981)97.

74 7 R.S. Nelson, G.P. Creissen and S.W.J. Bright, Plant Sci. Lett., 30 (1983) 355. 8 J.F. Shepard and R.E. Totten, Plant Physiol., 60 (1977) 313. 9 J.F. Shepard, Plant Sci. Lett., 18 (1980) 327. 10 J.F. Shepard, in: I. Rubenstein, B. Gengenbach R.L. Phillips and C.E. Green, (Eds.), Genetic Improvement of Crops: Emergent Techniques, Univ. of Minnesota Press, Minneapolis, 1980, p. 185. 11 J.F. Shepard, Plant Sci. Lett., 26 (1982) 127. 12 E. Thomas, Plant Sci. Lett., 23 (1981) 81.

13 M.A. Baer, S. Austin and rJ.P. Helgeson, Plant Physiol., 75 Suppl. (1984) 133. 14 S. Austin, M.A. Baer and J.P. Helgeson, Plant Sci., 39 (1985) 75. 15 G.T. Haberlach and J.P. Helgeson, Plant Physiol, 67 Suppl. (1981) 116. 16 J.P. Helgeson, B.A. Cohen, N.R. Hendricks, L.E. Towill and G.T. Haberlach, Plant Physiol., 69 Suppl. (1982) p 72. 17 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 963. 18 J.M. Widholm, Stain Technol., 47 (1972) 189.