Plant regeneration from adventitious roots of spinach (Spinacia oleracea L.) grown from protoplasts

ELSEVIER

Plant Science 120 (1996) 89 94

Plant regeneration from adventitious roots of spinach (Spinucicr oleracea L.) grown from protoplasts Fuminori

Komai”**, Kiyoshi “Department

hLahorator~~

of Horticulture.

of Hortictrltural

Masuda”, Hokkaido

and Agrictdtttral

Takashi

Lhicrrsit,v.

Sciences.

Sapporo.

Htroxaki

Harada”, Hokhtrido

(hliwrvit.\x.

Ichiro Okuseh MO. J~KIII

Hiroxrki.

.4rwmwi O.ih. Jrrpw

Received 22 March 1996; revised 14 June 1996: accepted 14 June 1996

Abstract An efficient system for plant regeneration from adventitious roots of spinach (Spinuc,itr &ruc~r L.) was established. The adventitious roots differentiated from calli that had grown from protoplasts. Protoplasts were obtained from the cotyledon of seedlings by partial digestion of cell walls, followed by mechanical maceration with forceps. The protoplasts formed colonies at a frequency of 1.6% in a Murashige and Skoog’s (MS) medium containing 1 LAM 2,4-dichlorophenoxyacetic acid (2,4-D) or 10 PM I-naphthalene acetic acid (NAA) combined with IO I_~Mh-(4-hydroxy-3-methyl-but-truns-2-enylamino) purine (zeatin). Calli derived from the colonies produced adventitious roots on MS medium containing IO PM NAA and 0.1 PM gibberellic acid (GA,). Root segments excised from these adventitious roots differentiated into many embryos when subcultured on MS medium supplemented with 30 mM fructose instead of sucrose. The embryos developed into flowering mature plants on a growth regulator-free medium. iG~~~orci.s: Plant regeneration:

Protoplast:

Somatic

embryogenesis:

Spinach

(.Spinuciu okm~u~w ): Tissue culture

1. Introduction

.4bhrrriations: BAP. 6-benzylaminopurine; 2.4-D. 2.4. dichlorophenoxyacetic acid; GA,, gibberellic acid; IAA. indole-3-acetic acid; IBA, indole-3-butyric acid: MES. ?-morpholino-ethanesulfonic acid: NAA, I-naphthalene acetic acid; Zeatin. 6-(4-hydroxy-3-methyl-but-trcrns-9-enylamino) purine. * Corresponding author, Hokkaido Prefectural Kitami Agricultural Experiment Station, Kunneppu, Hokkaido 09914. Japan. Fax: 81 157 47 2774.

In recent years, there has been a steady accumulation of molecular studies on the unisexual flower aimed at finding genes responsible for sex determination [I -51. Spinach, a dioecious species. has long been the subject of investigations into sex determination [6&8] using attempts to control sex expression under greenhouse[9] and tissue culture-conditions [IO]. Genetic manipulation using protoplasts may facilitate further investigations

0168.9452;96’$15.00 ‘FI 1996 Elsevier Science Ireland Ltd. All rights reserved PII

SO I68-9452(96)04463-9

90

F. Komai rt (11.:IPlant Scicwc~r 120 (1996) 89-94

into the cellular and molecular aspects of sex determination in spinach, because isolated protoplasts are available for cell fusion studies and genetic transformation. Protoplast and tissue culture techniques have proven useful for the analysis of sex expression in studies using the dioecious plant Melnndrizm dbunz [ 11,121. Spinach protoplasts have been isolated from leaves [13], plumule-derived calli [14], shoot apices [15], and cotyledons [16]. Goto and Miyazaki reported the shoot formation and plant regeneration from protoplast-derived calli of spinach [16]. However, the low plant regeneration frequency must be improved in order to apply these culture systems to other research purposes. Previously, we reported on the plant regeneration from root explants of spinach [17]. In the present paper, we describe an efficient procedure for plant regeneration from cotyledon protoplasts of spinach. The regeneration regime involves callus formation from protoplasts, adventitious root formation from the calli, and plant regeneration from the adventitious root segments. Morphological evidence that plant regeneration from root explants occurs through somatic embryogenesis is also presented.

2. Materials 2.1. Axenic

and methods culture qf’ spinuch seedings

Spinach (Spinucicr oleraceu L. cv. Jiromaru) seeds were surface-sterilized in 70% (v/v) ethanol for 30 s, and then in a sodium hypochlorite solution (3% chlorine) containing 0.1% Tween 20 for 2 h. After several rinses with sterile distilled water, the seeds were aseptically placed on Murashige and Skoog’s (MS) medium [18] with 20 g/l sucrose and 8 g/l agar. The seeds germinated and grew into seedlings at 25°C under a 16-h light/S-h dark photoperiod (40 uE m -’ s ~ ’ from cool white fluorescent tubes). 2.2. Isolation

of protophsts

Cotyledons, vested from

hypocotyls lo-day-old

and roots were harseedlings, while leaves

were harvested from 30-day-old plantlets. The tissues were incubated in an enzyme solution containing 2% (w/v) Cellulase ‘Onozuka’ R-10 and 0.5% (w/v) Macerozyme R-10 (both from Yakult Honsha Co. Ltd., Tokyo), in CPW9M/MES [19] comprised of CPW salts [20], 9% mannitol and 10 mM MES (pH 5.5). Approximately 1.0 g fresh weight of tissue was transferred to 10 ml of filtersterilized enzyme mixture solution without chopping into pieces and incubated at 25°C with reciprocal shaking at 70 rev./min in the dark. Since few protoplasts were released after incubation for 4-10 h, the tissue pieces were transferred to CPW9M/MES and gently squeezed with forceps to release protoplasts. After sieving the suspension through a 40-urn mesh screen, protoplasts were collected by centrifugation at 100 x g for 3 min. The protoplast pellet was resuspended in 15 ml of CPW9M/MES and washed 3 times as before. For further purification, the crude protoplasts were layered onto CPW salts containing 0.6 M sucrose and 10 mM MES (pH 5.5) in 115 x 15mm glass tubes and centrifuged at 100 x g for 3 min. The protoplasts which banded at the interface were collected and resuspended in 10 ml CPW9M/MES, followed by a wash in CPW9M/ MES. Viability of purified protoplasts was assessed by staining with fluorescein diacetate (FDA) [21]. -3.3. Protoplust

culture

The purified protoplasts were plated at a density of 1 x lob/ml in 60 x 15-mm plastic Petri dishes containing 2.4 ml medium consisting of half strength MS inorganic salts, MS vitamins with increased thiamine-HCl to 0.4 mg/l, 30 mM glucose, 0.46 M mannitol and 10 mM MES (pH 5.5) (colony formation medium). Auxins (30 uM IAA, 30 uM IBA. 10 uM NAA, or 1 uM 2,4-D) were added to the colony formation medium alone or in combination with either a cytokinin (zeatin or BAP at 0.1, 1 or 10 uM) or 0.1 uM GA,. Protoplasts were cultured in continuous darkness at 25°C for 3 weeks. The cultures were then diluted by adding 0.5 ml of the colony formation medium without mannitol every 3 days, and maintained at 25°C in a dim light (16-h day

F. Komai

et al.

/ Plant

length). After 3 weeks of the first dilution, the colonies that had developed to approximately 1 mm in diameter were transferred with forceps to the MS basal salts and vitamins as for colony formation medium supplemented with 30 mM glucose, 30 mM sucrose, 500 mg/l casein hydrolysate (Casamino Acids, Difco, Detroit, MI), 7 g/l agarose (Type II, Sigma, St. Louis, MO), 0.3 PM 2,4-D and 3 uM zeatin (pH 5.5) (callus formation medium). Cultures were kept at 25°C under a 16-h day length (40 pE m - 2 s - ‘). After 3 weeks of callus culture the calli were transferred to a regeneration medium which consisted of the same components as those of the callus formation medium, except that 10 pM NAA and 0.1 uM GA, replaced 2,4-D and zeatin. These calli formed somatic embryos and adventitious roots. Three weeks later, to promote organogenesis and embryogenesis, calli were transferred to the MS medium with 20 g/l sucrose and 7 g/l agarose (Type II, Sigma) but without any growth regulators. Plating efficiency was expressed as the percentage of protoplasts that produced multicellular colonies after 4 weeks of protoplast culture. All media used in the present experiments were sterilized by autoclaving at 120°C for 8 min. -3.4. Plant regeneration

from

adventitious

roots

Somatic embryos were produced from root segments according to the methods described previously Adventitious from roots V71. protoplast-derived calli were excised into 8-mm segments and cultured on semi-solid MS basal salts and vitamins as for colony formation medium containing 30 mM fructose [22], 7 g/l agarose (Type II, Sigma) and 10 uM NAA combined with/without 0.1 uM GA,. After 4 weeks, explants bearing embryos were transferred to the growth regulator-free MS medium for further development. Cultures were kept at 25°C under a 16-h day length. After 2 weeks, the number of embryos was counted under a dissecting microscope, and the frequencies of callus formation and embryogenesis were determined. Plantlets (5 - 10 mm in length) were transferred to the growth regulator-free MS medium which was divided into

Scienw

120 (1996)

89

‘) I

93

30 x 170-mm culture tubes. Cultures were maintained under the same conditions for somatic embryogenesis.

3. Results and discussion 3.1. Isolution

of spinach protoplasts

When protoplasts are isolated from leaf tissues, tissues are generally cut into small pieces to achieve more effective enzyme treatment. When this procedure was applied to isolate protoplasts from various types of spinach seedling tissue, almost all protoplasts remained in the tissues after incubation in the enzyme mixture, and most of the released protoplasts were damaged. Because of the difficulty in isolating protoplasts from spinach plants, we used a method [16] of mechanically breaking up tissues after partial digestion of cell walls. As a result, cotyledons consistently released healthy protoplasts with a yield exceeding lO’/g fresh weight of tissue (Table 1; Fig. 1A). Leaves also yielded a large number of protoplasts, but isolated protoplasts were less viable. Hypocotyls and roots yielded a smaller number of protoplasts (below 104/g of tissue) despite prolonged incubation and maceration with forceps. This procedure resulted in a sufficient yield of viable protoplasts for optimizing the culture conditions for cell division.

Cotyledon protoplasts were cultured in media containing several kinds of growth regulators to determine the effective combination of growth Table I Yields of viable explants (n = 4)

protoplasts

Explant

Protoplasts

Leaf Cotyledon Hypocotyl Root

1.46x107f0.17 1.42x107+0.14 4.33 X I@ & 0.57 I .52 X IO’ * 0.12

.‘Data

represent

from

different

(No.:@ tissue)”

the mean f S.D.

types

Viahilitv 47.3 87.7 33.6 ix.1

of spinach

(“I,),’

* Y.9 * 3.6 * 7.x & 5.5

Fig. 1. Somatic embryogenesis and organogenesis from calli of spinach (Spincrcia okracru L.) derived from cotyledon protoplasts. (A) Cotyledon protoplasts isolated from spinach seedlings. (B-D) Division of regenerated cells after 2 weeks of culture. (E) A typical cell cluster after 3 weeks of culture. (F) Visible colonies cultured in colony formation medium supplemented with I PM 2.4-D and 10 PM zeatin after 7 weeks of culture. (G) Protoplast-derived calli grown on callus formation medium supplemented with 0.3 FM 2.4-D and 3 FM zeatin. (H) Adventitious roots (arrows) differentiation from the calli. (I) Somatic embryogenesis from protoplast-derived callus. The bars represent 50 pm in (A) and (E) and 10 pm in (By-D).

regulators for colony formation (Table 2: Fig. lB-F). Since a single application of any growth regulator tested did not induce cell division of cotyledon protoplasts, combinations of growth regulators were required for sustained cell division. Combinations of 2,4-D and zeatin showed a higher plating efficiency than other combinations of growth regulators. When zeatin and BAP at 0.1, 1 or 10 FM were tested in combination with 30 FM IAA, 30 PM IBA, 10 PM NAA or 1 PM 2,4-D, protoplasts developed into colonies at 10 PM BAP in the presence of 2,4-D and 10 PM zeatin in the presence of NAA or 2,4-D. The highest value was scored at 10 PM zeatin in combination with 1 PM 2,4-D (Table 2). Follow-

ing the application of GA,, which promotes somatic embryogenesis in spinach, no cell division was observed from the protoplasts. 3.3. Enzbr~qyenesis

,fi;ovnprotoplast-derived

calli

When colonies raised from cotyledon protoplasts in colony formation medium containing 1 PM 2,4-D and 10 PM zeatin were subcultured in the same medium, the colonies grew into green calli exhibiting vigorous growth. In comparison, colonies transferred to the medium containing 10 PM NAA and 0.1 PM GA, grew into yellowwhitish calli and showed moderate growth. Colonies cultured on 2,4-D and zeatin-supple-

Table 2 Combinations of growth regulators used for colony from cotyledon protoplasts of spinach (n = 4) Growth

Auxin IOuM IOnM IO uM 10uM

Plating (“Y”),

regulators

formation

efficiency

Addenda NAA NAA NAA NAA

I uM 2,4-D 1 ).tM 2.4-D I gM 2,4-D I uM 2.4-D

0.1 uM GA, 10 uM BAP IO nM zeatin

0.0 0.0 0.0 0.1 kO.2

0.1 uM GA, 10 uM BAP IO pM zeatin

0.0 0.0 0.6 & 0.3 l.6+0.2

,‘Plating efficiency was recorded after 4 weeks culture. Data represent the mean + SD.

L. of protoplast

mented colony formation medium showed no signs of organogenesis or embryogenesis, while those cultured on the medium with NAA and GA, produced many adventitious roots and somatic embryos at an extremely low frequency (Fig. lG_I). Totally, 3 embryos were formed from 25 calli and 2 of them developed into plantlets. Prolonged culture exceeding 8 weeks on callus formation medium containing an auxin resulted in necrosis of the callus, and calli transferred to growth regulator-free medium failed to differentiate embryos. GA, inhibited cell division of cotyledon-derived protoplasts. Previously, we had demonstrated that GA, stimulated somatic embryogenesis from spinach root segments [23], and in the present experiments, it was observed that GA, promoted somatic embryogenesis from protoplast-derived calli (Fig. 11). Table 3 Somatic embryogenesis

from adventitious

Gro\cth regulators ~ _~__.___.

Callus

IO PM NAA 0.1 uM GA, 10 uM NAA+O.l

100 0 100

uM GA,

roots

formatiot+

a

Fig. 2. Regeneration of spinach plants from explants of adventitious roots which were produced by protoplast-derived calli. (A) An isolated embryo differentiated from adventitious root explants. (B) Developing plantlets on growth regulator-free medium. (C) Bolting and flowering of a protoplast-derived regenerant of spinach.

The calli that developed from protoplasts of spinach cotyledons produced many adventitious roots on callus formation medium containing 10 nM NAA combined with 0.1 PM GA, (Fig. 1H). As expected from previous results [17], explants excised from these adventitious roots generated many somatic embryos (Table 3; Fig. 3A). Combining NAA with GA, was efficient for somatic embryogenesis from the root segments of spinach. On the other hand, we optimized the conditions of growth regulators for root organogenesis from protoplast-derived calli. When IAA, IBA, or NAA at 10 or 30 PM were compared. adventitious roots were induced at the highest frequency (91.7%) from calli cultured on callus

of spinach (‘IL))

Embryogenesis”.’ 0.0 0.0 92.5

“Data based on 40 explants from two replicated experiments. ‘(No. of explants forming cdlli/No. of explants cultured) x 100. ‘(No. of explants producing embryos/No. of explants cultured) x 100

(“,tI)

No. of embryos 0.0 0.0 21.0 + 2.6

per explant

(mean + S.E.)

94

F. Komai et al. / Plant Science

formation medium containing 10 PM IBA (data not shown). Almost all of the somatic embryos germinated and developed into mature plants bearing flowers on the growth regulator-free medium (Fig. 2B and C). It was difficult to maintain a spinach callus retaining embryogenic potential. We previously reported continuous growth of excised roots of spinach in liquid culture [24]. The segments of adventitious roots grown in liquid culture exhibited a high embryogenic competence for more than 8 weeks of culture period (data not shown). Accordingly, the subculture of excised root tips may be applicable for the maintenance and proliferation of protoplast-derived spinach cell lines which are embryogenic. These studies demonstrated that plant regeneration from adventitious roots of spinach grown from protoplasts was possible. The established protoplast culture should facilitate cellular and molecular studies on sex expression and on crop improvement in spinach.

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