Bovine Haemoglobin Enhances Division of Plant Protoplast Cultures
(From Agricell Report 30(1):1-2. January 1998)

The inability to supply adequate amounts of oxygen to cells can limit the success of in vitro plant cultures. A recent method of improving oxygenation of plant cell or protoplast cultures involves the use of perfluorocarbons (PFCs) compounds that are remarkable for their ability to carry and release extremely large amounts of oxygen as compared with other liquids (J. Exp. Bot. 44(supplement):45, 1993; Agricell Report 21(1):1, 1993; Plant Cell Reports 13:251-255, 1994; J. Biotechnol. 50:377-393, 1996; U.S. Patent No. 5,180,676). At the University of Nottingham, P. Anthony, K.C. Lowe, M.R. Davey and J.B. Power have tested the ability of haemoglobin to enhance the supply of oxygen to, and improve division of cultured plant protoplasts, alone or in combination with PFCs (Biotechnology Techniques 11(8):581-584, 1997). As contrasted with PFCs, which are immiscible in aqueous systems, haemoglobin can be directly added to culture media. The immiscibility of PFCs, however, allows them to be reused, resulting in a cost savings as compared with haemoglobin, which has a limited shelf life.

Anthony et al. cultured Passiflora giberti and Petunia hybrida protoplasts in (1) liquid medium alone, (2) in liquid medium overlaying oxygenated perfluorodecalin (PFC), (3) in liquid medium containing a commercial bovine haemoglobin solution and (4) in liquid medium containing haemoglobin AND overlaying oxygenated perfluorodecalin. The University of Nottingham researchers found that while division of protoplasts derived from both plants was increased by all treatments, optimal results with Passiflora protoplasts were obtained with haemoglobin alone added to the medium (120% increase over liquid medium alone). In contrast, optimal division of Petunia protoplasts was achieved with the use of both oxygenated PFC and haemoglobin (140% increase over control).

The authors hypothesize that hemoglobin increases protoplast division by capturing dissolved oxygen from the air-medium interface and delivering it to the cells. According to Anthony et al., the results of their experiments suggest that, while the use of PFCs and haemoglobin provide new options for enhancing division of cultured plant cells, the relative effectiveness of oxygenated PFC, haemoglobin or combinations of the two may be species specific. They suggest that, for Passiflora, the combined treatment with PFC and hemoglobin may provide too much oxygen, resulting in oxygen toxicity.

The University of Nottingham investigators have also compared the ability of bovine haemoglobin to enhance division of P. hybrida protoplasts in the presence or absence of the copolymer surfactant Pluronic F-68 a compound that has been shown to promote growth of cultured plant cells, tissues and organs (Agricell Report 17(3):20, 1991: Agro-Food-Industry Hi-Tech 4:9-13, 1993).

Anthony et al. report that, after 9 days of culture, whereas haemoglobin alone resulted in a small but significant increase in Petunia protoplast plating efficiency, the combination of haemoglobin and Pluronic F-68 had a synergistic effect, resulting in a 92% increase in mean plating efficiency over that of untreated controls. In addition, the combined treatment produced a biomass increase 2.5-fold greater than that of the control over an 80-day period. According to the authors, haemoglobin "with or without Pluronic F-68, provides a further option for enhancing the growth of cultured plant protoplasts and protoplast-derived cells under static and, perhaps, agitated conditions." They add, "This technology could readily be scaled up to fermenter systems and may be effective in stimulating growth of cultured plant tissues and organs . . . which are now employed extensively as a source of economically important plant products."

For further information:
K.C. Lowe, Plant Genetics Manipulation Grp., Dept. of Life Sci., Univ. of Nottingham, University Pk., Nottingham, NG7 2RD, U.K. Fax: 115 951 3666.