Encyclopedia : Tissue culture & morphogenesis

Tissue culture, the adventive embryo formation and plantlet regeneration

Copyright 1998-2012 Yoshiaki Yoneda


Plant tissue culture began in the 1930s. This is a technique to culture excised cells, tissues and organs in glassware under controlled nourishment and physical conditions. When a hybrid embryo produced by interspecific crossings, for example, does not grow normally, the immature embryo can be removed and cultured in glassware with suitable nourishment, thereby producing a hybrid plant. Steward cultured tissue pieces of a root of a carrot in the 1950s, from which entire plants were successfully regenerated. Whole plant bodies have been regenerated from cultured tissues or cultured cells of many plants. Currently tissue culturing is utilized as one of the new breeding techniques.

In Ipomoea nil, when a section of stem or hypocotyls is cultured, a callus (amorphous tissue consisting of disorderly gathered cells) occurs and an adventitious root develops easily from this callus, but no adventitious bud occurs.

As a result of having examined various techniques to regenerate an individual body of Ipomoea nil in vitro, Yoneda, in cooperation with Mr. Nobuo Nakamura, developed a method to take out an immature embryo from a capsule 13 days after flowering and remove the cotyledons surgically (this is called a decotylated embryo) and culturing this decotylated embryo on agar medium containing inorganic salts with casamino acids and sugar. This result showed that many protuberances can occur on the tip of a hypocotyl of an immature embryo, and some protuberances are differentiated into adventitious embryos, and finally into entire plant bodies. Moreover, when the tip of the hypocotyls, including a radicle, was explanted as a conical body, adventitious embryos were found to grow rapidly on it (1987). This is the first individual regeneration process of Ipomoea nil. This method has since been improved. It is expected that completely new morning glory strains can be brought up if new genes are introduced using such an individual regeneration method.

Following are the references on tissue culture of Ipomoea nil. Individual regeneration of a sweet potato in genus Ipomoea was successful in the 1980s.


Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Callus and adventitious roots

When sections of a stem or a hypocotyl of Ipomoea nil were cultured on an agar nutrient basic medium including plant hormones, a callus occurred and an adventitious root occurred easily. Root-like structures were produced from the bottom of the callus shown in the Figure. However, differentiation of adventitious buds was not found.

Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Decotylated embryo culture --> Callus and various forms of protuberances

After removing the cotyledons surgically from the embryo of a cotyledon stage one week after fertilization and culturing a hypocotyl including a radicle, a callus occurred from a cut end, and projections of various shapes (tubes, cups, bars) arose from the border region of a callus and a hypocotyl. With this technique, you don't need to add any particular hormone to agar nutrient medium.

Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Hypocotyl end culture --> adventitious embryo

When the hypocotyl tip of an embryo at the cotyledonary stage one week after fertilization was cultured, an adventitious embryo occurred together with a projection of various shapes. Projections developed on the hypocotyl tip as shown in this figure, and an adventitious embryo appeared on the tip.

Asagao, Ipomoea nil ( Pharbitis nil ) Peking Tendan
Decotylated embryo culture --> regenerated plantlets developed from the adventitious embryo

In the regenerated plantlets that developed from an adventitious embryo, you can see a stem and a leaf.

Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Decotylated embryo culture --> a regenerated plantlet developed from the adventitious embryo

In the regenerated plantlets that developed from an adventitious embryo, you can see a stem, a leaf, and a root.

Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Decotylated embryo culture --> a regenerated plantlet developed from the adventitious embryo

The regenerated plantlets were transplanted into fairly big glasswares from test tubes.

Asagao, Ipomoea nil ( Pharbitis nil ) Tokyo Kokei Standard
Decotylated embryo culture --> transplant of the plant to a pot

The regenerated plantlets were transplanted into a flowerpot.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plantlet developed from adventitious embryo

The results were shown when a strain derived from the Violet strain was cultured by similar methods. A leaf, a stem, and a root were differentiated in a regenerated plantlet developed from an adventitious embryo.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plant

The regenerated plant transplanted to a glass bottle.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plant

The regenerated plant that flowered in a glass bottle.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plant

The regenerated plant transplanted into a flowerpot from a glass bottle.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plant

Flowering of the regenerated plant that was transplanted into a flowerpot from a glass bottle.

Asagao, Ipomoea nil ( Pharbitis nil ) 902M
Decotylated embryo culture --> a regenerated plant

A regenerated plant matured and opened double flowers.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Young embryo (at cotyledonary stage) about one week after fertilization

The left figure shows the embryo at the cotyledonary stage taken out from an ovule of closely related species Ipomoea hederacea. The right figure shows the tissue structure of the embryo at the cotyledonary stage in the embryo sac of an ovule.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo --> adventitious embryo formation

Cotyledon of the embryo at cotyledonary stage was surgically removed (this is called decotylated embryo). When this decotylated embryo was cultured, various forms of protuberances occurred.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo culture --> a regenerated plantlet

A plantlet developed from an adventitious embryo.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo culture --> a regenerated plantlet

The regenerated plantlet that was cultured and produced on nutrient basic medium with the addition of 55 mg/l glutaminic acid.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo culture --> transplant of the regenerated plant

Plantlet transplanted to a flowerpot from glassware. The plantlet was covered with a beaker for a while to avoid a drastic change of environment.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo culture --> a regenerated plant

The regenerated plant flowered in a flowerpot.

Amerikaasagao Ipomoea hederacea (= Pharbitis hederacea)
Decotylated embryo culture --> a regenerated plant

A further grown, flowering regenerated plant

A general commentary book of plant tissue culture

  1. Harada,H. and Komamine,A. (1979) Plant cell and tissue culture Practice/ application / prospects Rikougakusya.

References
  1. Butenko, R. G. (1964) Plant Tissue Culture and Plant Morphogenesis, 291pp. Academy of Sciences of the USSR (translated by M.Artmann,1968, Jerusalem).
  2. Matsubara, S. (1964) Abnormal organ formation from the hypocotyl of cultured young embryos in Pharbitis nil (preliminary report). Sci. Rep. Kyoto Prefectural Univ. (Natural Sci., Living Sci. Welfare Sci.) 15A: 15.
  3. Matsubara, S. and Nakahira, R. (1966) In Vitro formation of adventitious organs by Pharbitis embryos. Nature 2ll: 1208-1210.
  4. Harada, H. (1967) Flower induction in excised shoot apices of Pharbitis and Chrysanthemum cultured in vitro. Nature 214: 1027-1028.
  5. Bhar, D. S. (1970) ln vitro studies of floral shoot apices of Pharbitis nil. Canad. J. Bot. 48: 1355-1358.
  6. Minamitani, T. and Nakayama, I. (1970) Influence of many factors for Tadashi Minamitani / Nakayama being immense germfree culture flowering sprout. Miyazaki University Department of Education bulletin natural science 28: 6-20.
  7. Gunckel, J. E., Sharp, W. R., Williams, B. W., West, W. C. and Drinkwater, W. O. (1972) Root and shoot initiation in sweet potato explants as related to polarity and nutrient media variations, Bot. Gaz. 133: 254-262
  8. Messershmidt, M. (1974) Kallusbildung und Differenzieerung aus isolierten Protoplasten von Pharbitis nil. Z. Pflanzenphysiol. 74: 175-178.
  9. Rao, P. S. and Harada, H. (1974) Hormonal regulation of morhogenesis in organ cultures of Petunia inflata, Antirrhinum majos and Pharbitis nil. In: Plant Growth Substances l973, pp.1113-1120. Proc.8th Intl. Conf. Plant Growth Substances. Hirokawa Pub. Comp.,Tokyo.
  10. Sangwan, R. S. and Norreel, B. (1975) Pollen embryogenesis in Pharbitis nil L. Naturwissennschften 62: 440.
  11. Bapat, V. A. and Rao, R. S. (1977) Shoot apical meristem culture of Pharbitis nil. Plant Sci. Lett.10: 327-334.
  12. Sehgal, C.B. (1978) Regeneration of plants from anther cultures of sweet potato (Ipomoea batatas Poir.) Z. Pflanzenphysiol. 88: 349-352.
  13. Hisajima, S. and Thorpe, T. A. (1981) Lactose-adapted cultured cells of Japanese morning glory, Acta Physiol. Plant. 3: 187-191.
  14. Yoneda, Y. (1982) In vitro culture of immature embryos in morning glories. In: Plant Tissue Culture 1982 (Fujiwara, A., ed.), pp.779-780. Japanese Assoc. Plant Tissue Culture, Tokyo.
  15. Jarret, R. L., Salazar, S. and Fernandez, Z. (1984) Somatic embryogenesis in sweet potato HortScience 19: 397-398.
  16. Liu, J. R. and Cantliffe, J. (1984) Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomoea batatas Poir.) Plant Cell Reports 3: 112-115.
  17. Hisajima, S. and Thorpe, T. A. (1985) Carbohydrate utilization and activities of various various glycosidases in cultured Japanese morning-glory callus Plant Tissue Culture Lett. 2: 14-21.
  18. Yoneda, Y. and Nakamura, N. (1987) Embryoid formation and plantlet regeneration from cultured immature embryos in three strains of Pharbitis nil. Rep.Lib.Arts, Shizuoka Univ. (Sciences) 23: 11-20.s
  19. Yoneda, Y. (1990) Japanese morning glory In: Handbook of Plant Cell Culture 5, Ornamental Species (ed. by Ammirato,P.V. et al.) p.509 McGraw-Hill Publ. Comp., N.Y.
  20. Jia, S. and Chua, N. (1992) Somatic embryogenesis and plant regeneration from immature embryo culture of Pharbitis nil. Plant Science 87: 215.
  21. Otani, M. (1996) Application of plant biotechnology for breeding of Ipomoea species Bull. Ishikawa Agr. Coll. 26: 15-43.
  22. Otani, M. and Shimada, T. (1998) Embryogenic callus formation from immature embryo of Japanese morning glory(Pharbitis nil Choisy) Plant Biotechnology 15: 127-129.



Edited by Yuuji Tsukii (Lab. Biology, Science Research Center, Hosei University)