regeneration of fertile plants from isolated tobacco zygotes by in vitro culture

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810 Chinese Science Bulletin Vol. 49 No. 8 April 2004

Chinese Science Bulletin 2004 Vol. 49 No. 8 810 814

Regeneration of fertile plants from isolated tobacco zygotes by in vitro culture HE Yuchi, SUN Mengxiang & YANG Hongyuan Key Laboratory of MOE for Plant Developmental Biology, Wuhan Uni-versity, Wuhan 430072, China Correspondence should be addressed to Sun Mengxiang (e-mail: mxsun @whu.edu.cn)

Abstract Living zygotes of tobacco (Nicotiana TabacumL.) SR-1 were isolated and cultured in vitro by the microcul-ture technique. Fertile plants were regenerated from the calli derived from cultured zygotes via organogenesis. Ovules were collected 120 h after pollination and used as feeder. MS combined with KM8p was selected as basic medium in the experiment. Zygotes isolated from ovules 108 h after pollina-tion turned out to be suitable material for in vitro culture. Over 80% such zygotes could divide and around 10% of them could grow into calli and regenerate fertile plants. Keywords: tobacco, zygote, in vitro culture, plant regeneration.

DOI: 10.1360/03wc0605

In higher plants an egg cell fuses with a sperm cell and develops into an embryo, the new generation of sporophyte. Such a process deals with a series of complex and delicate developmental events, and has long been an attractive field to developmental biologists. However, the whole process happens deeply in ovules and is covered by various other plant tissues. It is difficult, therefore, to di-rectly observe and study the living developmental events of the process. If zygotic embryogenesis can be induced in vitro it will surely facilitate the study on developmental mechanism of embryogenesis and crop improvement. In fact, based on the in vitro embryogenesis system zygotic protoplasts can be useful material for gene transformation. Leduc et al.[1] introduced GUS and anthocyanin gene into maize (Zea mays L.) zygotes by microinjection and ob-served as high as 3.5% transient expression. Holm et al.[2]

also microinjected foreign genes into barley (Hordeum vulgare L.) zygotes. Transformed plants were regenerated from cultured barley zygotes. High percentage of transient expression of GFP was also gained by means of electro-poration [3,4]. The experiments indicate that zygotes culture combining gene transformation will offer a promising technique for both gene function analysis during embryo-genesis and crop improvement.

Since natural zygotes are deeply embedded in ovules it is difficult to isolate large amounts of zygotes and keep them growing in vitro. The success in zygotes culture de-pends on two key techniques: efficient isolation of viable zygotes and a qualified microculture system. The first

report on successful regeneration of fertile plants by zy-gotes culture was in maize[5]. Since then it has been suc-ceeded respectively in barley, wheat (Triticum aestivum L.) and rice (Oryza sativa L.)[6 10]. However, in dicotyledon no successful example has yet been reported up to now. We ever set up a method combining enzymatic digestion with mechanical grinding to isolate living zygotes and by coculturing with mesophyll protoplasts the isolated zy-gotes were induced to divide in vitro[11]. Later, up to 60% isolated zygotes could divide in vitro by improvement of culture condition, and some of the zygotes could further develop into embryos or microcalli[12]. Based on our pre-vious work we report in this paper the successful regen-eration of fertile plants from cultured zygotes in tobacco. 1 Materials and methods

( ) Materials. Three tobacco cultivars, SR-1, W38 and Gexing 1, were used for isolating zygotes. Plants were grown in a greenhouse at 25 , in 16 h light period.

( ) Methods for zygote isolation. Ovaries at dif-ferent stages after pollination were collected according to ovary length as reported by Fu et al.[11]. The ovaries were surface-sterilized rapidly by 75% ethanol, and then were sterilized again by 0.1% sodium hypochlorite for 5 min. 3 washes in sterile water were followed, each for 5 min. Fertilized embryo sacs were isolated first by enzymatic maceration and then by gentle grinding with a small glass pestle[11]. The zygotes were then mechanically isolated from the embryo sacs after second enzymatic treatment for 10 30 min with 0.25% Cellulase R-10 ( Yakul T Honsha Co., Ltd.) and 0.2% Macrozyme R-10 ( Yakul T Honsha Co., Ltd.) dissolved in 13% mannitol, pH 5.4. The microdissection was performed under an inverted micro-scope with hand-made micropipette.

( ) Preparation of feeder. Living ovules were used as feeder in this experiment. Ovaries were collected 120 h after pollination and surface-sterilized rapidly by 75% alcohol and then was put in 0.1% sodium hypochlorite for 5 min. 3 washes in sterile water were followed, each for 5 min. Ovules were further isolated from the ovaries and collected in 2 mL 13% mannitol. After being washed by 13% mannitol and culture medium the ovules were cul-tured in a 30 mm Petri dish, with 1.5 mL the same me-dium and at the density of 100 150/1.5 mL as a feeder system.

( ) Procedure of zygotes culture. The isolated zygotes were transferred into a Millicell (Millipore-CM, NO. PICM 012 50, diameter 12 mm) containing 100 Lmedium. The Millicell was previously placed in the Petri dish with 1.5 mL feeder. Five to eight zygotes were in-oculated in each Millicell. After culturing at 25 in the dark for 15 d, feeder was removed and multi-cellular structures were transferred to callus proliferation medium and cultured continuously at 25 in a rotary shaker at 25

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Chinese Science Bulletin Vol. 49 No. 8 April 2004 811

ration (Table 1).

r/min in the dark. Big calli were cultured on the medium for plant regeneration at 25 with 16 h light period. Plantlets were first grown in medium for strengthening before they were transplanted to soil.

( ) Media for zygotes culture. All liquid media were sterilized by filtration. Solid media were autoclaved before use.

(1) KM8p. KM8p basic medium[13] was supple-mented with 0.3 mol/L sucrose, 0.15 mol/L mannitol, 0.1 mol/L sorbitol, 10% coconut water, 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, pH 5.8.

(2) MS+B5. MS macroelements[14] combined with macroelement-removed B5

[15] were supplemented with 0.3 mol/L sucrose, 0.15 mol/L mannitol, 0.1 mol/L sorbitol, 10% coconut water, 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, pH 5.8.

(3) MS + KM8p. MS macroelements[14] combined with macroelement-removed KM8p[13] were supplemented with 0.3 mol/L sucrose, 0.15 mol/L mannitol, 0.1 mol/L sorbitol, 10% coconut water, 1.0 mg/L 2,4-D 0.5 mg/L 6-BA pH 5.8.

( ) Media for callus proliferation. The basic me-dia used for callus proliferation is the same as that used in zygote culture with some modifications. All liquid media were sterilized by filtration and solid media were auto-claved before use.

(1) MS + KM8p-1. MS + KM8p without coconut water was supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, 6.0% sucrose, pH 5.8.

(2) KM8p-1. KM8p basic components were sup-plemented with 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, 6.0% sucrose, pH 5.8.

(3) MS+B5-1. Macro-elements of MS and all ele-ments except for macro-elements of B5 were supple-mented with 1.0 mg/L 2,4-D, 0.5 mg/L 6-BA, 6.0% su-crose, pH 5.8.

( ) Medium for plant regeneration. MS-1, MS were supplemented with 1.0 mg/L 6-BA, 0.5 mg/L IAA, 3.0% sucrose, 0.8% agarose, pH 5.8.

( ) Medium for strengthening plantlets. MS-2, MS without phytohormone, 2.0% sucrose, 0.8% agarose, pH 5.8.

( ) Collection of data and images. Single cells and micro-calli were observed under an inverted micro-scope (LEICA DM IRB). Images were collected by

Cooled-CCD (CCD LAMBDA 10-2, Pioneer). Calli were observed and imaged by OLYMPUS SZX12 stereomicro-scope connected with the CCD. The image analysis was performed via MetaMorph software (Universal Imaging Corp).

( ) Data analysis. All experiments were repeated at least three times. Relevant data were calculated via Ex-cel software. 2 Results

( ) Genotype selection. There is no distinct dif-ference among the three tobacco cultivars used in our experiments concerning the time of the first zygotic division appearing. The first cleavage was usually observed 5 15 h after inoculation. Zygotes isolated from all of the three cultivars showed relatively high frequency of the first division. It was as high as 88.2% in SR-1 and even the lowest frequency in Gexing 1 could reach to 79.1%. However, the main differences were found in the potential of zygote continuing development. SR-1 zygotes were remarkably predominant in the frequency of multi-cellular structure formation, 44.4% of zygotes could develop into multi-cellular structures. Plantlets could only regenerate from the calli derived from SR-1 and W38. Generally, two or more plantlets could generate from the same piece of callus. Comparing with W38, SR-1 was superior concerning the developmental potentiality of zygotes and plant regene

( ) Basic medium selection. The comparative study among three basic media indicated that zygotes could divide almost at the same time in three media. The first division was observed 5 15 h after inoculation. The highest frequency of first division was 87.5% when zy-gotes were cultured in MS+KM8p, and it was 71.9% and 77.2% when in KM8p and MS+BB5 respectively. Such di-vision frequency is much higher than that of somatic pro-toplasts in the same culture medium, indicating that all three media could be used for triggering zygote division invitro. However, MS+KM8p showed more suitable for continuing zygotes development than the other two media. As high as 42.1% zygotes cultured in MS+KM8p could develop into multi-cellular structures and some of them could finally form big calli on proliferating medium (Ta-ble 2). On MS+B5 a few multi-cellular structures could be also found and develop into calli later. Plantlets could eas-

Table 1 Effect of genotype on the development of zygotes in vitroa)

Genotype Total no. of zygotes

Time of 1st division/h

Freq. of 1st division (%)

Freq. of multi-division (%)

Total no. of callus

Total no. of plantlets

SR-1 79 5 15 88.2 ± 4.0 44.4 ± 6.2 4 12W38 67 5 15 80.6 ± 3.5 13.3 ± 1.5 1 2

Gexing1 52 5 15 79.1 ± 4.9 26.3 ± 4.0 0 0

a) All zygotes were isolated from ovules 108 h after pollination. The medium for zygotes culture was MS+KM8p. Tobacco ovules isolated 120 h after pollination were used as feeder. The frequency of the first zygotic division and multi-division was calculated after 36 and 96 h culture respectively.

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Table 2 Effect of culture medium on zygotes development in vitroa)

Medium Total no. of zygotes


Freq. of 1st division (%)

Freq of multi-division (%)

Total no of callus


MS + KM8p 91 5 15 87.5 ± 3.1 42.1 ± 6.8 9 23KM8p 84 5 15 71.9 ± 4.0 29.5 ± 5.9 0 0

MS + B5 93 5 15 77.2 ± 7.1 31.4 ± 5.5 2 6a) All zygotes were isolated from ovules 108 h after pollination. The medium for zygotes culture was MS + KM8p. Tobacco ovules isolated 120 h

after pollination were used as feeder. The frequency of the first zygotic division and multi-division was calculated after 36 and 96 h culture respectively.

Table 3 Influence of zygotes stage on development in vitroa)

Zygotes stage Total no. of zygotes


Freq. of symmetricaldivision (%)

Freq. of asymmetricaldivision (%)

Freq. of multi-division (%)

Total no. of callus


72 h after pollination 51 24 48 33.9 ± 6.6 9.6 ± 3.7 11.1 ± 5.8 0 0108 h after pollination 73 5 15 10.9 ± 1.2 74.8 ± 7.8 43.1 ± 6.6 2 5

a) Zygote culture medium was MS+KM8p. Tobacco ovule isolated 120 h after pollination were used as feeder. Calli proliferation medium was MS+KM8p-1, differentiation medium was MS-1. The frequency of the first zygotic division and multi-division was calculated after 36 h and 96 h cul-ture respectively.

ily regenerate from the calli formed on both media. ( ) The effects of zygotes developmental stages on

their growth in vitro. Zygotes at different developmental stages were cultured in the same condition, the time, fre-quency and pattern of the first zygotic division were nota-bly different (Table 3). Zygotes isolated 72 h after pollina-tion commenced the first division slowly. It could be first observed at least 24 h after inoculation and most of zy-gotes divided after 36 48 h culture. The first division pattern of 33.9% zygotes appeared to be symmetrical and that of 9.6% zygotes was asymmetrical. By contrast, zy-gotes isolated 108 h after pollination could divide 5 15 h after being inoculated, and 74.8% of them divided asym-metrically and 10.9% of them divided symmetrically. With regard to the developmental fate of two daughter cells, one of the cells resulted from asymmetrical division showed dominant development, but two sister cells from symmetrical division usually hold the same developmental potentiality.

Only a few zygotes isolated 72 h after pollination could complete multi-division and form multi-cellular structures and most of multi-cellular structures were dis-organized and degenerated after 5 6 d culture. No big calli formed in this case. The first division frequency of zygotes isolated 108 h after pollination was very high, and nearly half of them could develop into irregular microcalli although only some compact microcalli was able to de-velop into big calli and regenerated into fertile plants via organogenesis.

( ) The developmental characteristics of zygotes in vitro. We traced the whole developmental process of the same zygotes in vitro, especially its early developmental stages (Fig. 1(a) (e)). The just isolated and inoculated zygote looked elliptic since the original cell wall materials was still partially attached on its surface (Fig. 1(a)). It completed the first unequal division after 5 h culture and the second division occurred after 24 h (Fig. 1(b)). Later

on, two different kinds of multi-cellular structures, com-pact and incompact structures, could be formed. Incom-pact multi-cellular structures consisted of loosely organ-ized cells. During their subsequent development the cyto-plasm in the cells gradually congregated around nucleus or cell wall and accumulated large black granules. This type of multi-cellular structures could maintain 4 5 d growth and then gradually became brown and disorgan-ized. The cells could lose all the contents in this case and only keep their empty cell walls. The compact type of multi-cellular structures consisted of cells in which cyto-plasm condensed but evenly distributed (Fig. 1(c)). Such structures could develop into bigger compact structures 3 d later, but none of them looked like an embryo morpho-logically (Fig. 1(d)). Hereafter, multi-cellular structures developed rapidly and formed compact microcalli after 10 d culture (Fig. 1(e)). At this time, the microcalli were moved onto fresh proliferation medium. They unceasingly grew up and formed bigger calli. The calli were then transferred on differentiation medium, MS-1, green buds extruded about 20 d later (Fig. 1(f)). Many buds could generate on the same piece of callus (Fig. 1(g)). Usually both bud and root differentiation could be induced on the same medium (Fig. 1(h)). When the regenerated plantlets were with 8 10 leaves they were strengthened on MS-2 that was free of any growth regulator and then transferred to soil (Fig. 1(i)). The plantlets finally developed into normal and fertile plants (Fig. 1(j) (k)). The germination frequency of seeds gained from the zygote-derived plants was about 95%. Their offspring looked normal and no obvious morphological variation was observed. It took 4 months around from inoculating zygotes to harvesting the seeds from the zygote-derived plants.

( ) Summary of suitable conditions for tobacco zy-gotes culture. According to the results of our experiment, the suitable conditions for tobacco zygotes culture can be summarized as follows: SR-1 zygotes isolated 108 h after

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Fig. 1. Fertile plant regenerated from an isolated tobacco zygotes by in vitro culture. (a)—(e) Images showing continuous developmental events of the same zygote. (a) A zygote freshly isolated 108 h after pollination, arrow indicates its nucleus; (b) a zygote divided two times after 24 h culture. Arrow indicates division plate; (c) multi-divisions of a zygote after 48 h culture; (d) multi-cellular structure derived from the zygote; (e) a compact mi-cro-callus grown from the structure in (d) 10 d later; (f) green buds (arrow) on callus; (g) many buds differentiated from the same piece of callus; (h) two plantlets regenerated from the same piece of callus; (i) the plantlets shown in (h) were transferred to soil and became vigorous plants after 15 d growth; (j) plant derived from the zygote developed normally and flowered; (k) the regenerated fertile plant.

pollination are inoculated on MS+KM8p liquid medium. The ovules of tobacco 120 h after pollination can be used as feeder. During this period 200 L MS+KM8p should be added to the petridish every other 3 d. After 15 d culture calli should be transferred to MS+KM8p-1 as proliferated medium and cultured in a rotary shaker at 25 r/min at 25 in the dark. Again, 200 L fresh MS+KM8p-1 was added to the petridish every other 3 d. After 10 d culture big calli should be transferred onto differentiation medium MS-1 under the light condition of 16 h/d. Regenerated

plantlets can now be moved to strengthening medium MS-2 and finally transferred to soil.

3 Discussion Tobacco zygotes has been successfully isolated many

years ago[11,12]; however, the zygotes culture has encoun-tered great difficulties although fertile plants have been successfully regenerated via zygotes culture in some of gramineous species, such as maize (Zea mays L.), barley (Hordeum vulgare L.), wheat (Triticum aestivum L.) and

Chinese Science Bulletin Vol. 49 No. 8 April 2004 813

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rice (Oryza sativa L.) in recent years[5 10]. The possible reason for this may deal with two aspects. One is that to-bacco zygotes isolation has to suffer long time of enzyme treatment and the prolonged enzyme digestion may have a negative effect on the viability and developmental poten-tiality of the isolated zygotes[12,16,17]. Such effects might be responsible also for the low frequency of plant regenera-tion in the present work. The other reason may be that there has been no efficient feeder used in tobacco zygotes culture. In prevenient experiments, suspension cells and precultured microspores were usually used as feeder[5 10].We chose ovules instead of mesophyll protoplasts that previously used as feeder in our experiment[11,12], which has greatly improved tobacco zygotes development in vitro. Zygotes grown in nature is surrounded by ovule tissues, which may provide necessary conditions for zy-gotes development and it was believed that they may also play the same role in zygotes culture. Present work indi-cates that the ovules are suitable feeder especially for maintain the continuing growth of zygotes in vitro, which is a main advantage compared to mesophyll protoplasts. Another advantage of the ovules as feeder is that every tobacco ovary possesses several thousands of ovules and therefore preparing this feeder system was comparatively simple and easy.

Rice zygotes culture showed distinct genotype effect on their development potential in vitro. Although there was no notable difference between Taipai and IR58 con-cerning the frequency of multi-cellular structure forma-tion that is 41.3% and 32.0% respectively, obvious dif-ference appeared in the frequency of plantlet regeneration. 36.2% zygotes of Taipai could grow into fertile plants, whereas only 4.0% of IR58 zygotes regenerated into nor-mal plantlets[10]. However, such genotype effect was not reported in barley, wheat and maize[5,8,9]. The results in the present work revealed that the first division of tobacco zygotes was independent of genotypes, but multi-cellular structure and calli formation were evidently affected by genotypes. SR-1 zygotes showed its obvious advantage in both cell division frequency and continual growth potenti-ality in vitro.

There are few reports up to now about the effects of zygote developmental stages on its development potential in vitro. Some researchers showed that zygote develop-ment in vitro was not influenced by its developmental stages in barley[8]. But in our previous experiment in to-bacco it was found that the division pattern and develop-mental fate, especially the time of the first division of zy-gotes largely depended on their developmental stages[12].This experiment suggested that zygotes at late stage are superior to that at early stages. This conclusion was in-consistent with the results from wheat zygotes culture[9],but it demonstrated again our previous observation in to-bacco zygotes culture[12].

Acknowledgements This work was supported by the National Natural Science Fund of China (Grant No. 30370743), the National Outstanding Youth Science Fund (Grant No. 30225006) and the State Key Basic Research and Development Plan of China (Grant No. 2002CCA00100).

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(Received December 11, 2003; accepted February 13, 2004)

regeneration of fertile plants from isolated tobacco zygotes by in vitro culture

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