Abstract:
Cassava (Manihot esculenta Crantz) is a vegetatively propagated root crop used
as a staple throughout the tropics and subtropics. It is the fourth most important
and cheapest staple food crop after rice, wheat and maize in developing
countries, providing food for over 600 million people. However, its production is
severely limited by a wide variety of viral and bacterial diseases, especially
Cassava Mosaic Disease (CMD) which is caused by several geminivirus species
including, South African cassava mosaic virus (SACMV), African cassava mosaic
virus (ACMV), East African cassava mosaic virus (EACMV), Indian cassava
mosaic virus (ICMV) and the Ugandan recombinant virus (UgV). In South Africa
(SA), there has recently been an enormous upsurge of interest in cassava for
industrial applications such as the manufacture of starch, animal feeds, and in its
potential as a food security crop for marginalised farmers. However, due to
serious losses in cassava yields by begomoviruses, such as SACMV, there is an
urgent need for the development of appropriate systems that allows for
transformation and regeneration of virus-resistant transgenic cassava cultivars
suitable for diverse needs and growth requirements in different geographical
areas in southern Africa.
The potential application of cassava tuber disks as an alternative system
to leaf tissue for transformation and regeneration was investigated. Furthermore,
the antibiotic, carbenicillin, was tested as a possible shoot inducing factor. Disks
from freshly-harvested cassava tubers were cultured on 25 different sets of MS
supplemented with zeatin (0.01-5 mgl-1) and indole-3-acetic acid (0.01-5 mgl-1).
Carbenicillin at 500 μgl-1 was included in each treatment as a potential
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organogenesis inducing factor. The results observed after 21 days in culture
indicated that non-embryogenic friable callus formed readily on MS medium
supplemented with MS vitamins, 30 gl-1 sucrose, 0.01 mgl-1 indole-3-acetic acid
(IAA), 0.01 mgl-1 zeatin (ZEA), 500 μgml-1 carbenicillin and 0.8% agar, pH 5.8.
Shoots or somatic embryos were never formed and only adventitious roots
developed at a frequency of 60% on shoot induction medium supplemented with
2μM copper sulphate (CuSO4), 1 mgl-1 6-benzylaminopurine (BAP) and 0.5 mg-1
indole-3-butyric acid (IBA).
The current study also investigated infection of cassava and tobacco by
the SA begomovirus species SACMV, dimer A and B using the particle inflow
gun. Full-length head-to-tail dimers of DNA-A and DNA-B of SACMV were
constructed by digestion with SalI or EcoRI, respectively. The DNA-coated
particles were used to shoot 3-week-old cassava plantlets (cv. TMS60444) at a
pressure of 1500 psi using the Bio-Rad biolistic device. Thirty-day-old N.
benthamiana seedlings were also inoculated in the same manner. In both cases
young tender uppermost leaves were targeted (five plants inoculated and another
5 as control). Disease symptoms were recorded daily on the first emerging
leaves. Cassava plantlets and tobacco seedlings showed infection by visibility of
symptoms. On the other hand, control plantlets that were not inoculated were
symptomless. Symptoms appeared 7 dpi in tobacco whereas mosaic symptoms
became visible 14 dpi in cassava.
The pre-requisite for any cassava transformation program that proposes to
develop improved plants is the availability of a reliable regeneration system.
Presently many laboratories that prioritize cassava research are able to reliably
