Transcript Extending Cassava shelf-life through exploring Induced Mutations

Extending cassava (Manihot esculenta) shelf-life through exploiting
induced mutations
Olalekan Akinbo1, Emmanuel Okogbenin1, Chiedozie Egesi1, Chikelu Mba2, Favour Ewa1,
Oluwakemi Ogundapo1, Kenneth Eluwa1, Eunice Ekaette1, Lydia Ezewanka1, John Beeching3 and
Martin Fregene1
1National Root Crops Research Institute (NRCRI), P.M.B 7006, Umuahia, Abia State, Nigeria; 2International Atomic
Energy Agency, Vienna, Austria; 3University of Bath, United Kingdom; 4Donald Danforth Plant Science Center, 975 N.
Warson Rd. St Louis, MO63132, USA
Corresponding author E-mail: [email protected]
Abstract
A
Cassava (Manihot esculenta) roots spoils within 48-72 hours after harvest due to postharvest physiological
deterioration (PPD) which accounts for significant annual yield losses globally. Cassava is allogamous and
highly heterozygous showing high segregation at F1 for several key breeding traits. Unfortunately, the
development of delayed PPD varieties has been hampered due to lack of genetic variation in available breeding
gene pools. Mutation induction is a proven technology for creating and improving genetic variability in a crop
variety. In this study, initiatives were undertaken to use induced mutation to create and breed for delayed PPD
in cassava. Two high yielding elite varieties (TMS95/0379 and TMS98/0002) known for resistance to disease
and pest were selected as planting materials for induced mutation to develop delayed post harvest deterioration
tolerance in cassava. These materials were irradiated at 12 and 15 gamma ray level as in vitro cultures at the
International Atomic Energy Agency (IAEA), Vienna, Austria. They were micro-propagated through several
cycles to reduce chimera. They were subjected to hardening process and transferred to the field for evaluation
for mutants for delayed PPD at the National Root Crops Research Institute (NRCRI), Umudike, Nigeria. Yield
and delayed PPD were evaluated at 7 and 14 days after harvest (DAH). Over 2000 M1 cassava plants were
initially planted on the field, however, only 50% of the M1 plants showed good establishment resulting in the
evaluation of about 824 M1 genotypes for delayed PPD and other important agronomic traits such as yield, dry
matter content and harvest index. Phenotypic variation for PPD and other traits were observed within M1
derivatives of the same parental clone suggesting possible genetic variations due to mutation. Similar
observation was made for other traits. Traits correlations were highly significant between PPD and other
evaluated traits. Results indicate that nine M1 plants showed no physiological post harvest deterioration
representing 1% of the evaluated M1 population.
Introduction
Results
Post harvest physiological deterioration (PPD) as expected was significantly higher at 14 DAH than at 7DAH.
While PPD was fairly similar at 7 DAH for both irradiation levels, significant difference was observed between
14 DAH for the two dosage level with higher PPD at 15 GY than at 12GY indicating that 12GY resulted in
lower PPD in the M1 population. Results showed wide variation PPD with most of the M1 genotypes showing
high PPD. The histogram for PPD distribution at 14 DAH in the M1 population is shown in Fig 1. Results
indicate that nine M1 genotypes showed no PPD and possibly representing new mutants for this trait. M1
derivatives from parental clone TMS 95/0379 had six of the genotypes with zero PPD while the other three are
M1 genotypes of TMS 98/0002. The result of the PPD evaluation of the M1 genotypes and the control
genotypes are shown in Fig. 2. The control for both 12 GY and 15 GY were 80% deteriorated after 14 days.
Post harvest physiological deterioration for 7 days after harvest in 12 GY irradiated genotypes was
B
significantly correlated with 14 days after harvest
P≤0.0001 (Tables 1). PPD correlation patterns with most of
C
the traits were similar for the two PPD evaluation dates (7 and 14 DAH). Results showed that PPD either at 7
DAH or 14 DAH was not significantly correlated with fresh root yield, dry root yield, dry matter content and
harvest index either at 12 GY in the M1 population. However, at 15 GY, PPD was significantly correlated to
dry root yield (7 DAH) and harvest index (14 DAH).
50
30
Cassava deterioration (PPD) is an active process involving changes in gene expression and protein synthesis,
together with the accumulation and oxidation of phenolic compounds, that shows parallels with normal plant
wound responses but in which wound repair is inadequate and the resultant return to normal development
absent (Reilly et al., 2007). The exclusion of oxygen through waxing the roots on harvest prevents PPD, which
is a viable solution to PPD for expatriate consumers in developed countries prepared to pay a premium for their
traditional food, but is not an economic solution to ordinary consumers for such a low value crop.
Conventional breeding has been used successfully to improve cassava quality with non polygenic traits, but it
remains a problematic tool due to the highly heterozygous nature of the crop, inbreeding depression and
variable fertility (Kawano, 2003). However, the recent identification of cassava variants and hybrids with high
delayed PPD suggests that breeding should not be completely discarded as a potential solution to controlling
PPD (Morante et al., 2010). The ability to induce stable mutations in plant tissues and seeds followed by the
identification and selection of useful mutant material opens up an alternative avenue to the improvement of
cassava with respect to its PPD response that avoids many of the difficulties and challenges associated with
breeding and genetic modification. We report here results from induced mutation studies on the development
of delayed PPD mutant in cassava and its significance for cassava.
40
No of Genotypes
No of Genotypes
25
20
15
10
30
20
10
5
0
0
0
20
40
60
80
100
0
20
40
60
80
100
% PPD 14 DAH
% PPD 14 DAH
Fig. 1. Histogram of PPD evaluation at14 days after harvest for a set of M1 genotypes at 12 GY and 15 GY irradiation dosage level
Materials and Methods
Two released elite varieties (TMS 95/0379 and TMS 98/0002) were subjected to 12 and 15 gamma ray
irradiation. Both varieties are resistant to cassava mosaic disease with good yield and dry matter content. The
irradiated in vitro cultures were brought in from International Atomic Energy Agent (IAEA), plantlets remained
in the growth chamber for six weeks before they were transferred to the green house under protection from
direct sun and insects, the plants were hardened and then transplanted in the field at National Root Crops
Research Institute, Umudike, Nigeria. The irradiated in vitro was planted four times to guarantee material from
those genotypes that may not survive the hardening and field transplanting in July 2009. The field layout was
1 m x 1 m between and within rows for the 5 plants stand replicated thrice. The plantlets were arranged
genotype by genotype on the field. In the field, rainfall served as source of water supply.
Primary deterioration of cassava results from an endogenous physiological process, independent of the
presence of pathogens, and has been described as a wound response cascade gone awry (Wheatley, 1982).
Three to seven roots were randomly picked per genotype and used to determine post harvest physiological
deterioration in individuals of TMS 98/0002 and TMS 95/0379 of 12GY and 15GY level of irradiation.
Evaluation for PPD was done at seven and fourteen days after harvest. Immediately after harvest, 10 - 15 cm
sections were taken from each randomly picked root (Marriot et al., 1978). The distal or tail end of the cut roots
was covered with a PVC film to prevent water loss. The proximal cut surface was exposed to low humidity
conditions for seven and fourteen days, the first time interval routinely used for PPD evaluation. At seven and
fourteen days after harvest, ten transverse sections were made at 2 cm intervals from the proximal end of the
root section. Physiological deterioration manifests as discolouration of the vascular tissues and storage
parenchyma. The extent of the vascular discolouration is a measurement of the susceptibility of the genotype
to PPD. Each of the seven sections was scored on a scale of 0 to 10 (where 0 = 0% deterioration, 2 = 20%
deterioration and 9 = 90% deterioration) and average percentage deterioration determination for each genotype.
Sigmaplot 10.0 (2007) statistical programmes were used for correlation, and frequency distributions of
phenotypic classes.
Table 1. Phenotypic correlation for irradiated 12GY and 15GY for yield and post harvest
physiological deteriorations
12GY
15GY
FRY (ton ha-1)
12GY
15GY
DRY (ton ha-1)
12GY
15GY
DMC (%)
12GY
HI (0-1)
7DAH (%)
0.21 ns
0.01ns 0.18*
14DAH (%)
0.15ns
0.01ns 0.13ns 0.05ns -0.02ns 0.04ns 0.34*
aFresh
15GY
12GY
15GY
7DAH (%)
-0.03ns -0.04ns 0.13ns 0.08ns -0.08 ns
-0.14 ns 0.49****
root yield; bDry root yield; cDry matter content; dHarvest Index; eDay after harvest
Fig 2. Response of the roots in the irradiated TMS 98/0002 and TMS 95/0379 with the
un-irradiated clones evaluated on the 14DAH
Discussion
Mutation breeding is an established method of crop improvement and has played a major role in the
development of mutant varieties (Datta and Chakrabarty, 2009). The lack of delayed PPD in available cassava
gene pools has been a major constraint in breeding to reduce losses. Complex problems associated with
marketability from short shelf life of the roots have necessitated the need to explore induced mutagenesis for
genetic improvement of the crop for this trait. Results indicate that nine M1 genotypes were identified as
showing delayed post harvest deterioration after two years of evaluation. In a recent publication, three
genotypes were found to show zero PPD (Morante et al., 2010). Mutation is often generally recessive and
masked at the M1 in inbred lines in the heterozygous state until fully expressed at M2 when mutant alleles are
fixed in the homozygous state.
Six of the nine genotypes showing zero PPD were mainly from the parental clone TMS 95/0379. The TMS
95/0379 is a variety with yellow root which is associated with pro-vitamin A - the precursor of beta carotene.
Reports by Morante et al (2010) indicate that high catenoid content in roots tended to have lower incidence of
PPD. It has been suggested that the antioxidant properties of carotenoids may help delay or prevent the
occurrence of oxidative process associated with delayed PPD (Reilly et al., 2003). This may also have
contributed to strong expression of delayed PPD in the mutants. Although nine genotypes were found to show
zero PPD, the M1 genotypes were micro-propagated after irradiation to reduce chimeras through several cycles
of multiplication. It is possible that some of the identified genotypes of the same parental clone may be
replicated copies genetically.
The discovery of zero PPD in nine putative mutants from this study provides a huge opportunity for these
genetic resources to be explored further in the breeding programme for the development of new varieties for
novel traits presently limited in gene pools.
Acknowledgements
We acknowledge the part funding received from International Atomic Energy Agency on the project
0.32*
References
Datta SK and Chakrabarty D. 2009. Management of Chimera and In vitro mutagenesis for
development of new flower/shape and chlorophyll variegated mutants in Chrysanthemum.
In Q.Y. (ed). Induced Plant Mutations in the Genomics Era. FAO. Pp. 303-305.
Kawano K. 2003. Thirty years of cassava breeding for productivity - Biological and social
factors for success. Crop Science, 43: 1325-1335.
Marriot J, Been BO, Perkins C. 1978. The aetiology of vascular discoloration in cassava
roots after harvesting: association with water loss from wounds. Physiologia Plantarum 44:
38-42.
Morante N, Sanchez T, Ceballos H, Calle F, Perez JC, Egesi C, Cuambe CE, Escobar AF,
Ortiz D, Chavez AL, Fregene M. 2010. Tolerance to Postharvest Physiological Deterioration
in Cassava Roots. Crop Science, 50: 1333-1338.
Reilly K, Bernal D, Cortes DF, Gomez-Vasquez R, Tohme J, Beeching JR. 2007. Towards
identifying the full set of genes expressed during cassava post-harvest physiological
deterioration. Plant Molecular Biology, 64: 187-203.
Reilly K, Gómez-Vásquez R, Buschmann H, Tohme J, Beeching JR. 2003. Oxidative stress
responses during cassava post-harvest physiological deterioration. Plant Molecular Biology,
53: 669-685.
Wheatley, CC. 1982. Studies on Cassava (Manihot esculenta Crantz) Root Post-Harvest
Physiological Deterioration. Ph.D. Thesis, University of London, 246p.