Plant regeneration from stem-derived protoplasts of Brassica alboglabra bailey

Plant Science, 50 (1987) 153 160 Elsewer Smentlfic Pubhshers Ireland Ltd

153

P L A N T R E G E N E R A T I O N FROM S T E M - D E R I V E D P R O T O P L A S T S OF B R A S S I C A ALBOGLABRA BAILEY

ENG-CHONG PUA* Laboratory of Plant Molecular Bmlogy, The Rockefeller Unwers~ty, 1230 York Avenue, New York, NY 10021-6399(U S A ) (Received October 24th, 1986) (Rewsmn received January 21st, 1987) (Accepted January 27th, 1987) Protoplasts isolated from stem tissue of Brass~ca alboglabra Bailey divided rapidly and formed mlcrocalh on medium supplemented with 0 2 mg/1 dlchlorophenoxyacetlc acid (2,4-D) and 0 2 mg/1 6-benzyladenme (BA) On shoot regeneratmn medium, the presence of 0 25 I mg/1 BA was most effective m inducing shoot and plant regeneration from protoplast-demved callus at a frequency of 18 and 24%, respectlvely While low cytokmm concentratmns enhanced rhlzogenesls, callus growth was promoted in the presence of 2,4-D or naphthalene acetic acid (NAA) All 20 regenerated plants were successfully acchmatlzed of which 19 were diploid and one was aneuplold Key words Brass~ca alboglabra, Chinese kale, plant regeneration, protoplast culture, t~ssue culture

Introduction The u t l h z a t m n of p l a n t p r o t o p l a s t s for gene t r a n s f e r w a s o m a t i c h y b r l d l z a t m n [1] or direct u p t a k e of n a k e d D N A [2,3] or t u m o r - r e d u c i n g (TI) plasmld v e c t o r [4], has a g r e a t p o t e n t m l for crop i m p r o v e m e n t A m e t h o d by w h i c h p l a n t s c a n be r e g e n e r a t e d from p r o t o p l a s t s ~s i m p o r t a n t for the p r o d u c t m n of g e n e t i c a l l y modified p l a n t s derived either by somatic hyb r l d l z a t l e n or cell t r a n s f o r m a t m n The g e n u s Brass~ca includes a wide r a n g e of crop species w~th g r e a t e c o n o m i c v a l u e worldwide P l a n t s h a v e been r e g e n e r a t e d from different g e n o t y p e s of Brass~ca i n c l u d i n g B napus

*Present address Institute of Molecular and Cell Biology, The National Umverslty of Singapore, Kent Ridge, 0511, Repubhc of Singapore Abbreviations BA, 6-benzyladenme, CGI, callus growth mdex, 2,4-D, 2,4-dlchlorophenoxyacetlc acid, 21p, 6-7,/dlmethylallylammo purme, IAA, mdole-3-acetlc acid, IBA, 3-mdolebutyrlc acid, MES, 2-(N-morphohno)ethanesulfomc acid, MS, Murashlge and Skoog, NAA, naphthalene acetic acid, PC, protoplast culture, R, rooting, SG, seed germination, SI, shoot mductmn, SR, shoot regeneratmn, TI, tumor-inducing

[5,8], B oleracea [9,10] and B j u n c e a [11] In addition, s o m a t i c hybmds were g e n e r a t e d as a result of the successful p r o t o p l a s t fusion bet w e e n B oleracea and B c a m p e s t n s [12], and b e t w e e n R a p h a n u s s a t w u s and B napus, and R s a t w u s and B c a m p e s t n s [13]. P a s z k o w s k l et al [14] r e c e n t l y r e p o r t e d successful genetic t r a n s f o r m a t i o n of B campestr~s v a r rapa protoplasts, but no plants were r e g e n e r a t e d from the t r a n s f o r m e d cells B alboglabra (Chinese kale) is an import a n t vegetable crop m E a s t e r n Asia P r e v i o u s studies showed t h a t p l a n t s could be regenerated from c o t y l e d o n o u s and h y p o c o t y l explants of this species [15,16] However, p l a n t regenera t l o n from p r o t o p l a s t s has n o t yet been rep o r t e d This s t u d y descmbes p r o c e d u r e s for the isolation, c u l t u r e and p l a n t r e g e n e r a t i o n from stem-demved p r o t o p l a s t s of B alboglabra

Materials and methods P l a n t materials Seeds of B alboglabra Barley (yellowflowered Chinese kale, from Sunmse Enterprise, Elmwood, CT) were surface-stemhzed by

0618-9452/87/$03 50 ~(, 1987 Elsevier Scientific Pubhshers Ireland Ltd Pubhshed and Printed m Ireland

154

dipping into 95% (v/v) ethanol for 10-15 s followed by continuous a g l t a t m n for 30 mln in 20% (v/v) commercml bleach (Clorox ~) solutmn (1% (w/v) sodmm hypochlorlte final concentratmn), rinsed twice w~th sterile &stilled water and germinated m SG medmm (Table I). Plant cultures were also m l t m t e d by transferring shoot cuttings to rooting (R) medmm (Table I) on which they were rooted and maret a m e d for 6 weeks In all the experiments reported here, the constituents (except zeatm) were added to the medmm and the pH adjusted to 5.8 before autoclawng at 1 kg cm ~ and 121°C for 20 min. Zeatm was filter-sterilized and added to the medmm after autoclawng. Cultures were maintained under a 16-h hght/8-h dark cycle with a photon fluence rate of about 1 5 0 p E m - 2 " s -~ (380800 nm) from Sylvania GRO-Lux F96T12/Gro fluorescent lamps The temperatures were 27 + I°C and 23 +_ I°C m the h g h t and dark, respectively.

Protoplast ~solatmn Stem segments (about 5 mm m length) from

T a b l e I. C o m p o m t l o n o f v a r m u s m e d m u s e d for c u l t u r i n g p r o t o p l a s t s a n d t i s s u e s of B alboglabra Constituent

mg/1

M S . m a c r oa

MS-m,cro b

myo-Inosltol Vitamin mixture c Dffco-bacto a g a r Sucrose Glucose NAA IBA BA

100 8000 10 000 30 000 20 000 60 000 1 01 2

Medmm SG

SI

PC

SR

R

+ + + ÷ +

+ + + + +

+ + + +

+ + + + + +

+ + + + +

+

+

+ + -t-

+ +

a M S [17] m a c r o e l e m e n t s , e x c e p t FeSO4"THsO a n d Na2E D T A w e r e r e p l a c e d by 40 mg/1 F e N a 2 - E D T A (13% Fe) (J T B a k e r C h e m i c a l Co ) b M S [17] m m r o e l e m e n t s c T h e m i x t u r e y m l d e d a final c o n c e n t r a t i o n of 0 1 mg/1 nlcot m l c acid, 0 1 mg/1 p y r l d o x m e - H C 1 , 0 01 rag/1 t h m m m e HC1 a n d 0 4 rag/1 g l y c m e

3-week-old plant cultures were grown on shoot induction (SI) medium (Table I) for 10-14 days before using them for protoplast isolation Freshly excised cotyledons and hypocotyls from 6-day-old seedhngs, and young leaves from 3-week-old plant cultures were also used as sources of protoplasts Explants were cut into small pmces (0.5-1 mm), and transferred to a Petrl dish with 20-25 ml of filter-stemlized enzyme solution containing CPW salts (1 5 g/l CaC12"2H20,. 246mg/1MgSO4"7HsO, 101mg/ 1 K N Q , 27.2 mg/1 KH2PO4, 0 16 mg/1 KI and 0.025 mg/1 CuSO4"5H20), 5 mM 2-(N-morphohno)ethanesulfomc acid (MES), 9% (w/v) manmtol, 1% (w/v) cellulase Onozuka R-10 and 0 2% (w/v) Macerozyme R-10 (Yakult Pharmaceutical Industry Co., Nishmomlya, Japan) The tissues were incubated for 4-5 h at room temperature (25_+2°C) with continuous agitation at 5~60 rev./min on a rotary shaker The digestion mixture was filtered through a 60#~m nylon sieve and the filtrate was centrifuged at about 100 × g for 6 mm The pellet was suspended m CPW salts containmg 21% (w/v) sucrose and the floated protoplasts were collected and washed twice with CPW salts containing 9% (w/v) glucose The protoplasts were plated at a denmty of 5 × 10~ protoplasts/ml in a 60 × 15ram Petrl dish containing 4ml of protoplast culture (PC) medium (Table I) supplemented with various combmatmns of 2,4-D, NAA and BA (see Results sectmn).

Protoplast culture and plant regeneratmn Freshly isolated protoplasts grown on various media were kept in the dark at room temperature for 4 days and then were cultured under the conditions described in the Plant Materials section. After 7 days the cell division frequency (number of divided cells per 200 cells counted) was determined m duphcate. Cells were transferred to 100 × 20 mm Petrl dishes, 10 ml of fresh medium was added after 7 days and 5 ml of medmm was added at weekly intervals thereafter For reduction of shoot regeneratmn, mlcrocalh demved from stem protoplasts grown on a

155

medmm containing 0.2 mg/1 2,4-D and 0.2 rag/1 BA were used Mlcrocalh were washed twice with seed germmatmn (SG) medmm before transfer to shoot regeneratmn (SR) medmm (Table I) m the absence of growth regulators or m the presence of (1) BA, 6-?,7-dlmethylallylammo purme (21p), zeatln or k m e t m , each at concentratmns of 0 125, 0.25, 0 5, 1, 2 and 4 rag/ 1, and (n) B A m combination with lndole-3acetm acid (IAA), 3-mdolebutyrlc acid (IBA), NAA or 2,4-D, at a concentratmn of 0 25 mg/1. Mmrocalh used for (1) and (n) were 5- and 6-7week-old, respectively Regeneratmn frequency (number of calh forming shoots and/or roots as a percentage of the total number of calh) was determined 3 weeks after plating Callus growth was measured using a callus growth index (CGI) m which the numemcal values 0, 1, 2 and 3 represent, respectively, dead or no growth, poor, good and excellent growth of the callus

Rooting and plant acchmat~zatmn Regenerated shoots, 10 mm or longer, were transferred to Plantcon ~-~ (Flow Laboratomes, Inc.) containing R medmm (Table I) for rooting and the rooted shoots were acchmat~zed by

transferring to Jiffy-7 peat pellets (Jiffy Products L t d , Shlppegan, Canada) moistened with half strength Murashlge and Skoog (MS) mineral salts Plants directly regenerated from callus were transferred to peat pellets without passing through the rooting stage After 5 days, all acchmanzed plants were transferred to 15 mm dmmeter plastic pots each containing Metromlx ~, a commercial greenhouse soil Plants were grown in the greenhouse and a nutment supplementation of 20N" 20P 20K was apphed at weekly intervals

Chromosome number determ~natmn After 3 weeks, root tips of 20 acchmatlzed plants were excised for chromosome number determination using the squash method [18] The tissues were pre-treated with 0 1% colchlcme for 2-3 h, fixed m a mixture of 95% ethanol/glacial acetm acid (3 1, v/v) for 24-48 h at room temperature, and stained with fermc aceto-carmme (1 ml of fermc acetate was added to 500 ml of aceto-carmme). Results Stem tmsues of B alboglabra grown on SI medmm (Table I) after 10-14 days enlarged

T a b l e II. Effect of g r o w t h r e g u l a t o r s on dlvmlon frequency of p r o t o p l a s t s from v a r i o u s explants of B alboglabra after 7 days m c u l t u r e 2,4-D (mg/I)

NAA (mg/1)

BA (mg/1)

Source of p r o t o p l a s t s a Cotyledon

0 0 0 0 0 02 02 1 1 1

02 02 1 1 1 0 0 0 0 0

0 02 0 02 04 0 02 0 02 04

5 5 2 4 2 27 35 20 18 12

Hypocotyl Leaf (division frequency 10 11 6 10 7 42 77 30 28 20

4 6 3 3 2 22 28 16 15 10

Stem

%)b 12 10 14 17 12 55 82 37 30 21

All e x p l a n t s were u n t r e a t e d except t h a t stems were g r o w n on SI m e d m m (Table I) for 10 14 days h The frequency was calculated based on the n u m b e r of divided cells m a total of 200 cells counted E a c h value r e p r e s e n t s the mean of two r e p h c a t e s

156

-{

~ AJ ,s

/,

$

C

J

~L

157

Both stem- and hypocotyl-derlved protoplasts behaved similarly m culture. The first (Fig. 1B) and second (Fig. 1C) cell division occurred 36 and 60 h, respectively, after isolation. After 10 days an 8-cell stage was observed (Fig. 1D) and after 5-6 weeks cells grew into yellowlsh and light green microcalli occasionally accompanied by globular orgamzed structures (Fig. 1E). Some microcalli floated to the surface of the medium. Mmrocalli originating from stem protoplasts were transferred to SR medium (Table I) containing different growth regulators for induction of shoot regeneration, prior to 8 weeks in culture since prolonged culture ( > 8 weeks) resulted in their discoloration. A comparative study on shoot regeneration from hypocotyl protoplast-derived mmrocalli was not conducted due to culture

considerably and abundant green, nodule-like structures formed at both cut ends of the tissues. The nodule-like structures developed into adventitious shoots after prolonged culture ( > 3 weeks). Cotyledon- and leaf-derived protoplasts grew slowly on all media tested (Table II). Browning began to occur 8-12 days after lsolatmn and cell division was arrested after 14-18 days. In contrast, protoplasts isolated from stem (Fig. 1A) and hypocotyl tissues grew equally well, particularly in the presence of 2,4-D (Table II). While the highest growth rate m terms of cell dlvismn frequency occurred on medmm supplemented with both 0.2 mg/1 2,4-D and 0.2mg/1 BA, growth was considerably slower m the absence of 2,4-D or in the presence of NAA (Table II). T a b e l III. Cytokmm

Effect of c y t o k m m on organogenesls frequency of stem protoplast-derlved callus of B alboglabra Organogenesls

Concentration (mg/1) 0

0 125

0 25

05 1 2 (organogenesls frequency %)a

4

0 35 0

0 15 1

13 6 2

18 10 4

15 5 4

8 2 0

5 1 0

BA

Shoot Root Shoot + root

21p

Shoot Root Shoot + root

0 32 0

0 12 0

5 9 0

9 11 0

8 10 0

0 4 0

Zeatln

Shoot Root Shoot + root

4 24 0

5 8 0

0 0 0

0 0 0

0 0 0

0 0 0

Kmetm

Shoot Root Shoot + root

0 38 0

0 22 0

1 5 0

0 0 0

0 0 0

0 0 0

a The frequency was calculated based on the number of call1 that formed shoots and/or roots as a percentage of the total number of calh after 2 weeks m SR medmm (Table I) Each value represents the mean of two rephcates

Fig. 1. Isolation, culture and regeneration of plants from stem-der,ved protoplasts of B alboglabra (A) Freshly isolated protoplasts grown on PC medmm (Table I) containing 0 2 mg/1 2,4-D and 0.2 mg/1 BA (400 × ), (B) 2-cell stage (400 × ), (C) 4-cell stage (400 × ), (D) 8-cell stage (400 × ), (E) mmrocallus with an orgamzed, globular-like structure (100 × ), (F) shoots and plants regenerated from callus grown on SR medmm (Table I) containing 0 25 rag/1 BA (2 × ), and (G) protoplast-derlved plants acchmatlzed and grown m the greenhouse for 4 weeks (0 1 x )

158 Table IV. Effect of BA with or without auxin on growth and organogenesls of stem protoplast-denved callus of B alboglabra after 2 weeks m culture Growth regulatora

CGIb

Organogenesls

BA BA + IAA BA + IBA BA + NAA BA + 2 4-D

05 0 0 25 30

Shoots, roots Root hairs Root hairs

a Each growth regulator was used at a concentration of 0 25 mg/1 m SR medmm (Table I) b Callus growth index 0, 1, 2 and 3 represent, respectively, dead or no growth, poor, good and excellent growth of the callus Each value represents the mean of two rephcates c o n t a m m a t m n H o w e v e r , a p r e h m m a r y lnvest~gatmn showed t h a t h y p o c o t y l protoplastderived m l c r o c a l l i g r o w n on SR medium c o n t a i n i n g 0.5 rag/1 BA formed shoots at a freq u e n c y of 11%. The g r o w t h p a t t e r n of stem protoplastderived m l c r o c a l h m terms of o r g a n o g e n e s l s and CGI is g r e a t l y influenced by the p r e s e n c e of g r o w t h r e g u l a t o r s m the c u l t u r e medium. Of all c y t o k m m s tested (BA, 21p, k m e t i n and zeatm), BA, p a r t i c u l a r l y at c o n c e n t r a t m n s of 0.25-1 rag/l, was most effective for s h o o t and p l a n t r e g e n e r a t i o n at a f r e q u e n c y of 18 and 24%, r e s p e c t i v e l y ( T a b l e III; Fig. 1F) On the o t h e r hand, r o o t f o r m a t m n o c c u r r e d freq u e n t l y at low c y t o k m l n c o n c e n t r a t m n s (0.125-0.25 mg/1) Callus grew p o o r l y on m e d m m c o n t a i n i n g BA with or w i t h o u t IAA or IBA, as evidenced by low CGI values (Table IV). In c o n t r a s t , callus a c c o m p a m e d by r o o t hairs grew w g o r o u s l y m the p r e s e n c e of BA in c o m b m a t m n with e i t h e r 2,4-D or NAA All r e g e n e r a t e d shoots o r i g i n a t i n g from stem p r o t o p l a s t s g r o w n m R m e d m m formed roots after 10 days B o t h types of plants (4 reg e n e r a t e d plants and 16 r o o t e d shoots) were successfully a c c h m a t l z e d in the g r e e n h o u s e (Fig 1G) N i n e t e e n p r o t o p l a s t - d e n v e d plants had a diploid c h r o m o s o m e n u m b e r of 18 and were p h e n o t y p l c a l l y m d l s t m g m s h a b l e from seed-derived plants, w h e r e a s only one p l a n t was a n e u p l o l d (2n = 15) and showed a r e d u c e d g r o w t h r a t e and smaller and distorted leaves.

Discussion P l a n t s h a v e been r e g e n e r a t e d from protoplasts isolated from various explants of Brass~ca i n c l u d i n g leaves [6,7], c o t y l e d o n s [5], h y p o c o t y l s [8,19], roots [20], and stem embryos [21] H o w e v e r , the type of tissue used as a s o u r c e of p r o t o p l a s t s is an i m p o r t a n t f a c t o r affecting p r o t o p l a s t p r o h f e r a t l o n and subseq u e n t p l a n t r e g e n e r a t m n . The h y p o c o t y l s gave c o m p a r a t i v e l y h i g h e r yields of p r o t o p l a s t s w h m h u n d e r w e n t dlvismn at h i g h e r frequencies, m c o m p a r i s o n with mesophyll p r o t o p l a s t s [8]. B r o w n i n g f r e q u e n t l y o c c u r r e d m slowgrowing mesophyll p r o t o p l a s t - d e r l v e d cells of Brass~ca [8,22]. It was suggested t h a t slowgrowing cells but not rapidly growing cells of Brass~ca p r o d u c e d toxic substances, p r o b a b l y p h e n o h c compounds, w h i c h inhibited cell g r o w t h [8,22] This can p r o b a b l y explain the b r o w n i n g and the s u b s e q u e n t g r o w t h mhlblt m n t h a t o c c u r r e d in slowly growing cotyledon- and leaf-derived protoplasts, but not m rapidly growing stem- and h y p o c o t y l - d e n v e d p r o t o p l a s t s of B alboglabra Nevertheless, this study md~cates t h a t a jud~cmus selection of d o n o r tissue is ~mportant to o b t a i n viable protoplasts which can divide e a r l y after l s o l a t m n at high frequency. Cell g r o w t h m w t r o is m a r k e d l y affected by g r o w t h r e g u l a t o r s m the c u l t u r e m e d m m Of all g r o w t h r e g u l a t o r s , 2,4-D, NAA a n d / o r BA h a v e been most f r e q u e n t l y used for c u l t u r i n g Brass~ca protoplasts [23] Results of th~s study are m a g r e e m e n t w~th p r e w o u s findings m w h m h 2,4-D p r o m o t e d g r o w t h of protoplasts and callus [8,24]. H o w e v e r , p l a n t r e g e n e r a t i o n of B. alboglabra was inhibited m the p r e s e n c e of 2,4-D or o t h e r a u x i n s A l t h o u g h c y t o k m m m c o m b m a t m n with a u x i n was r e p o r t e d to be beneficml to p l a n t r e g e n e r a t m n of v a r m u s Brass~ca g e n o t y p e s [8,19], this study demonstrates t h a t c y t o k i n m alone, p a r t i c u l a r l y BA, was most effective m the m d u c t m n of s h o o t a n d / o r p l a n t r e g e n e r a t m n of B alboglabra On the o t h e r hand, rhizogenems from protoplastderived callus of B alboglabra was e n h a n c e d m the absence of c y t o k m m or m the p r e s e n c e of low c y t o k m m c o n c e n t r a t m n s This result,

159 together with the finding that shoot cuttings of B. alboglabra r o o t e d r e a d i l y m t h e a b s e n c e of a u x i n ( u n p u b l i s h e d ) , i n d i c a t e t h a t cells of this genotype probably contain a relatively h i g h l e v e l of e n d o g e n o u s a u x i n . Phenotyplc and cytogenetlc varmtmn a m o n g p l a n t s r e g e n e r a t e d from p r o t o p l a s t s has been well documented m varmus plant s p e c m s i n c l u d i n g Solanum tuberosum [25-27] a n d Brass~ca spp. [8,28] I n Brass~ca s p p , morphologmal differences were detected among p r o t o p l a s t - d e r ~ v e d p l a n t s , a n d s o m e s h o w e d red u c e d g r o w t h a n d f e r t l h t y [8] R o b e r t s o n a n d E a r l e [10] r e p o r t e d a r e l a t i v e l y h i g h f r e q u e n c y (11.4%) of p o l y c o t y l e d o n o u s s e e d h n g s from t h e s e e d p r o g e n y of p r o t o p l a s t - d e r l v e d p l a n t s of B. oIeracea C y t o g e n e t l c a n a l y s i s of 25 plants r e g e n e r a t e d from mesophyll protoplasts of d i p l o i d B napus s h o w e d t h a t 44% of t h e plants were diploid and the remaining were a n e u p l o l d , i n c l u d i n g 20% h y p o d l p l o l d a n d 36% h y p o t e t r a p l o l d [28]. I n c o n t r a s t , t h i s s t u d y s h o w s t h a t t h e m a j o r i t y of t h e p l a n t s (95%) d e r i v e d f r o m s t e m p r o t o p l a s t s w e r e d i p l o i d Th~s h i g h f r e q u e n c y of n o r m a l r e g e n e r a t e d p l a n t s f r o m p r o t o p l a s t s offers a n a l t e r n a t i v e a p p r o a c h for m l c r o p r o p a g a t m n of B alboglabra I n a d d i t i o n , t h e c u l t u r e d cells m a y be u s e f u l for g e r m p l a s m p r e s e r v a t m n This study demonstrates a high frequency of cell d l w s m n l e a d i n g to s u b s e q u e n t p l a n t reg e n e r a t i o n f r o m s t e m - d e r i v e d p r o t o p l a s t s of B alboglabra T h e m e t h o d u s e d w a s s i m p l e a n d r a p i d W h o l e p l a n t s f r o m p r o t o p l a s t s w e r e obt a i n e d w i t h i n 12 w e e k s . V a r i o u s s p e c m s of Brass~ca i n c l u d i n g B alboglabra h a v e r a p i d r e p r o d u c t i v e c y c l e s [29]. T h e a b i l i t y to r e g e n erate n o r m a l plants from protoplasts will m a k e it p o s s i b l e to t r a n s f e r g e n e s v m a T1 p l a s m l d i n t o B alboglabra for p o s s i b l e c r o p improvement

Acknowledgments T h e a u t h o r w i s h e s to t h a n k Dr T r m h T h l T o a n H a n h for h e r a d v i c e , D r N a m - H m C h u a for h i s c r l t m a l r e a d i n g a n d c o m m e n t s on t h e manuscript, and Wendy Rome and Mrs Teresa Seow for skillfully typing the

m a n u s c m p t E.C.P. is a R e s e a r c h F e l l o w on l e a v e from t h e I n s t i t u t e of M o l e c u l a r a n d Cell B i o l o g y , T h e N a t i o n a l U n i v e r s i t y of S i n g a pore, K e n t Ridge, S i n g a p o r e 0511

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