Plants regenerated from mesophyll protoplasts of white mulberry

Abstract

Morus alba(white mulberry) mesophyll protoplasts were isolated from leaves of 30-45 day old sterile shoots, with protoplast yields of 2.5 × 107 g−1/F.W. after purification. The protoplasts were cultured in a modified K8P liquid medium containing 0.2 mg/L 2,4-D(2,4- Dichlorophenoxy acetic acid), 1 mg/L NAA(Naphthyl acetic acid) and 0.5 mg/L BA(6-benzylaminopurine). A low plating density (5 × 104/ml) proved to be favourable to the division of protoplast-derived cells. The first division occurred 4 days after culture, and the division frequency reached 24% at 10 days. A number of cell colonies and microcalli formed in 6 weeks. The microcalli were transferred onto MSB medium with 0.5 mg/L NAA and 0.5 mg/L BA for further proliferation. Shoot forrgation was initiated when the calli of 3-4 mm in size were transferred onto MSB differentiation medium with 0.1 mg/L NAA and 1 mg/L BA. The frequency of shoot formation was 35%. The shoots of 4-5 cm in height were excised from the callus and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA. After transplantation into pots, the regenerated plants grew vigorously in the phytotron.

Introduction

Protoplast culture is one of the most rapidly developing areas in plant tissue culture, because of its importance in plant genetic manipulation. However, so far, there are only a few forest tree species in which plant regeneration from protoplasts has been successful, namely Liriodendron tulipifera1, Paulownia fortunei2, Picea glauca3, P1atanus orientalis4, Populus sp.5, 6, 7, 8, Santalum album9, Solanum dulcamara10 and Ulnus1. White mulberry ( Morus alba) is an important woody feed crop for the silkworm. Some tissue culture has been carried out with this species, in an attempt to provide artificial feed containing cultured mulberry cells and for clonal propagation (see Review of Oka and Ohyama, 12). In this paper, we report plant regeneration from mesophyll protoplasts of this species.

Materials and Methods

Plant material

White mulberry seeds, collected in our institute, were surface-sterilized by dipping in 70% alcohol for 30 sec and then soaking in a sodium hypochlorite solution (containing 0.5% of effective chlorine) for 15 min. After being washed with sterile water (4 changes), the sterilized seeds germinated on hormone-free MS medium13. Apical buds cut from the seedlings were cultured on MS medium supplemented with 0.2 mg /L BA (at 25 °C; 2000 Lux, 12 h/ day) for inducing formation of multiple shoots. The multiple shoots formed were subcultured once a month for micropropagation of the plant material.

Protoplast isolation and culture

Young expanded leaves, collected from 30-45 day old cultured shoots, were cut into 0.5-1 mm strips and plasmolyzed for 1.5-2 h in CPW-11M (containing 11% mannitol). For releasing protoplasts, the leaf strips were digested statically in an enzyme solution for 4 h and then on a shaker (35 rpm) for 1 h. The enzyme solution was consisted of 1% Cellulase Onozuka R-10, Macerozyme R -10 and 0.05% Pectoltase Y -23 in CPW -9M (containing 9% mannitol), pH 5.7. The protoplasts in enzyme solution were filtered through an 45 μm nylon sieve and collected by centrifuging at 800 rpm for 4 min. They were purified by using a routine procedure employing floatation in CPW- 18S (containing 18% sucrose) and 3 washes in CPW -9M solution (centrifuged at 1000 rpm for 3 min). The purified mesophyll protoplasts were counted and protoplast viability was examined with 0.1% FDA in 0.35 mol/ L glucose solution.

The mesophyll protoplasts were cultured in 6 cm plastic dishes, each containing 2 ml K8P liquid medium14, supplemented with 0.2 mg /L 2,4-D, 1 mg /L NAA and 0.5 mg /L BA, at densities of 1×104, 5 × 104, 1×105, 5× 105, 1 ×106 and 5×106 protoplasts / ml to compare the effect of plating density on the division of the protoplast- derived cells. The dishes were placed at 25±1°C in dark. Division frequency was counted at 10 day. After small colonies formed (about 10 d), the dishes were moved under light (about 500 lux), and the cultures were diluted at 10 d intervals with 0.5 ml K8 medium14 with 0.1 mg /L 2,4-D, 1 mg /L NAA and 0.2 mg /L BA, for sustaining cell divisions and further stimulating cell colony formation and growth. Plating efficiency (% of number of small calli and cell colonies formed in the total number of the protoplasts cultured) was counted after 5-6 weeks of culture when small calli of 0.5–1.0 mm in size formed.

Callus proliferation

The small calli of 0.5-1.0 mm in size were transferred onto MSB growth medium (containing MS minerals13 and B5 organic components15) supplemented with 0.1–0.5 mg/L NAA and 0.25–1.0 mg/L BA for further growth and proliferation. The culture conditions were 25 ± 1°C with a 12 h photoperiod at 1000 Lux.

Plant regeneration

After 2 weeks on MSB growth medium, the calli grew up to 3-4 mm in size. Compact and light yellow calli were picked out and transferred onto MSB differentiation media with different types and concentrations of hormones for shoot formation. Shoots of 4-5 cm in height were cut off and rooted on half strength MS medium with 0.5 mg/L IBA and 0.1 mg/L BA. Regenerated plants were transplanted into pots, and grown in the phytotron of our institute.

Results and Discussion

Mulberry protoplasts were readily isolated from young expanded leaves of sterile shoots (Fig 1, 2), with a high yield of 2.5 ×107g/F.W. after purification. FDA fluorescent staining showed that the viability of isolated protoplasts reached 96.8%. Pretreatment of the leaf strips in CPW-11M for plasmolysis reduced the time required for enzymatic digestion. Incubation in enzyme solution, firstly in stationary and then on a shaker, caused less damage to protoplasts and was beneficial to maintain protoplast survival.

Fig 1
figure1

Protoplast culture and plant regeneration of white mulberry.Viability of freshly isolated mesophyll protoplasts of white mulberry was examined with FDA (fluorescein diacetate) staining. Viable protoplasts made up 96.8% of the population. × 400

Fig 2
figure2

Protoplast culture and plant regeneration of white mulberry.Freshly isolated mesophyll protoplasts from sterile shoots. × 400

The data listed in Tab 1 showed that protoplast density in culture apparently affected the divisions of the protoplast-derived cells. Within range of 1×104-5 106 /ml, the best results of time needed to initiate the first division and division frequency was from the group of 5 × 104 /ml protoplast density. In this case, the first division was observed at 4 d (Fig 3), the second division at 7 d (Fig 4), with cell clusters at 12 d (Fig 5). Higher densities (5×105, 1×106, 5 × 106) were unsuitable for the initiation of division and its maintenance for colony formation. It was demonstrated that mulberry mesophyll protoplasts could be induced to divide and form colonies when they were cultured in a thin layer of liquid medium at lower density. A number of small calli of 0.5–1.0 mm in size formed after 6 weeks of culture (Fig 6).

Tab 1 Effect of protoplast densities on division frequency
Fig 3
figure3

Protoplast culture and plant regeneration of white mulberry.The first division occurred after 4 d of culture. × 400

Fig 4
figure4

Protoplast culture and plant regeneration of white mulberry.The second division occurred after 7 d of culture. × 400

Fig 5
figure5

Protoplast culture and plant regeneration of white mulberry.Cell colony formed at 12 d in protoplast culture, × 100

Fig 6
figure6

Protoplast culture and plant regeneration of white mulberry.Protoplast -derived calli in 60 × 15 mm plastic Petri dish, after 6 weeks of culture in K8p liquid medium with 0.2 mg /L 2, 4-D, 1 mg /L NAA and 0.5 mg /L BA.

Hormone components in both the proliferation medium and the differentiation medium remarkably affected shoot regeneration from protoplast-derived calli (Tab 2 and 3). When the small calli of 0.5–1.0 mm were transferred onto MSB proliferation medium with 0.25–0.5 mg/L NAA and 0.25-1 mg/L BA, compact and light-yellow calli with a potential for shoot formation were formed (Fig 7), From the data in Tab 2, the best results in shoot regeneration was obtained in the calli cultured on the proliferation medium with 0.5 mg/L NAA and 0.5 mg/L BA. In order to test the effect of hormone component and concentration in differentiation medium on shoot formation, the calli of 3-4 mm in size cultured on the proliferation medium with 0.5 mg/L NAA and 0.5 mg/L BA were transferred onto MSB media supplemented with 0.1 mg/L NAA and different concentrations of BA or ZT (zeatin). The data in Tab 3 show that the differentiation medium with 0.1 mg/L NAA and 1 mg/L BA gave the best result. Green spots appeared on the calli 2-3 weeks after transfer onto this medium, followed by bud formation through organogenesis (Fig 8). The frequency of shoot formation reached 35%. When green shoots of 4-5 cm in height were cut off and transferred onto half strength MS rooting medium, vigorously growing roots formed and plants were produced 2 weeks later (Fig 9). Some of the regenerated plants were transplanted into pots, with survival rate of more than 90%. They grew into trees with 10-14 leaves, in the Phytotron of our institute (Fig 10).

Tab 2 Effects of hormone components in proliferation medium on shoot regeneration from mesophyll protoplast-derived calli of muiberry
Tab 3 Effects of hormone components in differentiation medium on shoot regeneration from mesophyll protoplast-derived calli of mulberry
Fig 7
figure7

Protoplast culture and plant regeneration of white mulberry.Compact and light- yellow calli formed on MSB proliferation medium with 0.5 mg/ L NAA and 0.5 mg /L B A.

Fig 8
figure8

Protoplast culture and plant regeneration of white mulberry.Shoot formation on MSB differentiation medium with 0.1 mg /L NAA and 1 mg /L BA.

Fig 9
figure9

Protoplast culture and plant regeneration of white mulberry.Complete plant regenerated after rooting on half strength MS medium with 0.5 mg /L IBA and 0.1 mg /L BA.

Fig 10
figure10

Protoplast culture and plant regeneration of white mulberry.The regenerated mulberry plant grown in pot.

To our knowledge, this is the first report on protoplast culture and plant regeneration in white mulberry. The results obtained showed that the expanded leaves from shoot culture were excellent material for protoplast isolation and culture for this tree. The controlled environment made the cultured shoots easily available and uniform, with a very high yield of protoplast. The mesophyll protoplasts from the cultured shoots were readily induced to divide and had a good potential of plant regeneration. Besides, the procedure we developed also gave high reproducibility in experiments. Now it is possible to improve this important tree species through gene transfer by using protoplasts as recipient.

References

  1. 1

    Merkle SA, Sommer HE . Regeneration of Liriodendron tulipifera (Family Magnoliaceae) from protoplast culture. Amer J Bot 1987; 74:1317–21.

    Article  Google Scholar 

  2. 2

    Wei ZM, Kamada H, Harada H . Plant regeneration from leaf mesophyll protoplasts of Paulownia fortunei. Plant Tissue Culture Letters (Japan) 1991; 8:110–3.

    Article  Google Scholar 

  3. 3

    Attree SM, Dunstan DI, Fowke LC . Plantlet regeneration from embryogenic protoplasts of white spruce (Picea glauca). Bio / Technology 1989; 7:1060–2.

    Google Scholar 

  4. 4

    Wei ZM, Xu ZH, Xu N, Huang MR . Mesophyll protoplast culture and plant regeneration of oriental planetree (Platanus orientalis). Acta Bot Sinica 1991; 33:813–8.

    CAS  Google Scholar 

  5. 5

    Russell JA, McCown BH . Culture and regeneration of Populus leaf protoplasts isolated from non-seedlong tissue. Plant Science 1986; 46:133–42.

    Article  Google Scholar 

  6. 6

    Russell JA, McCown BH . Recovery of plants from leaf protoplasts of hybrid- popular and aspen clones. Plant Cell Reports 1988; 7: 59–62.

    CAS  PubMed  Article  Google Scholar 

  7. 7

    Wang SP, Xu ZH, Wei ZM . Culture and regeneration of popular mesophyll protoplasts. Science in China(Ser.B) 1991; 34:587–92.

    CAS  Google Scholar 

  8. 8

    Park YG, Son SH . In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid popular (Populus nigra X P. maximowiczii). Plant Cell Reports 1992; 11:2–6.

    CAS  PubMed  Article  Google Scholar 

  9. 9

    Rad PS, Ozias-Akins P . Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.). Protoplasma 1985; 124:80–6.

    Article  Google Scholar 

  10. 10

    Chand PK, Davey MR, Power JB . Efficient plant regeneration from cell suspension protoplasts of the woody medicinal Solanum dulcamara L. (bitter-sweet, woody nightshade). Plant Cell Tissue & Organ Culture 1990; 22:119–25.

    Article  Google Scholar 

  11. 11

    Sticklen MB, Domir SC, Limeberger RD . Shoot regeneration from protoplasts of U lmus X 'Pioneer'. Plant Science 1986; 47:29–34.

    CAS  Article  Google Scholar 

  12. 12

    Oka S, Ohyama K . Mulberry (Morus alba L.). In: Biotechnology in Agriculture and Forestry (Ed. Y.P.S.Bajaj), Springer-Verlag, 1986; 1:384–93.

  13. 13

    Murashige T, Skoog F . A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 1962; 15:473–97.

    CAS  Article  Google Scholar 

  14. 14

    Kao KN . Chromosomal behaviour in somatic hybrids of soybean-Nicotiana glauca. Mol Gen Genet 1977; 150:225–30.

    Article  Google Scholar 

  15. 15

    Gamborg OL, Miller RA, Ojima K . Nutrient requirements of suspension culture of soybean root cells. Exp Cell Res 1968; 50:151–8.

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

This research work was supported by the National Biotechnology Programme and the Biotechnology Projects of the Ministry of Forestry (1991-1995).

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Wei, Z., Xu, Z., Huang, J. et al. Plants regenerated from mesophyll protoplasts of white mulberry. Cell Res 4, 183–189 (1994). https://doi.org/10.1038/cr.1994.19

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Keywords

  • Morus alba L.
  • white mulberry
  • protoplast culture
  • plant regeneration

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