Cell Biology And Morphogenesis

Plant Cell Rep (2002) 21:226–232 DOI 10.1007/s00299-002-0516-2

CELL BIOLOGY AND MORPHOGENESIS

K.E. Danso · B.V. Ford-Lloyd

Induction of high-frequency somatic embryos in cassava for cryopreservation

Received: 25 January 2002 / Revised: 1 August 2002 / Accepted: 10 August 2002 / Published online: 17 September 2002 © Springer-Verlag 2002

Abstract Methods for inducing high-frequency somatic embryos in cassava on cotyledons and 33 clonal accessions by the addition of supplementary copper sulphate to the induction medium were investigated. The addition of copper sulphate enhanced primary embryo induction and significantly increased secondary embryo production. All accessions from Latin America (CIAT) were embryogenically competent on medium supplemented with 8 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1 µM copper sulphate as were 15 of the 18 accessions from Africa. The percentage of calli producing somatic embryos ranged from 7.5% in M. Bra 12 to 100% in M. Col. 1505, while the number of embryos produced per callus ranged from 0.3 in M. Bra 383 to 13.5 in TEK. The frequency of embryo production was dependent on the concentration of copper sulphate. The number of primary embryos produced per callus was also comparatively higher in the medium supplemented with copper sulphate than in the controls. The optimal concentration of copper sulphate for number of embryos produced in most accessions was 5 µM, and at this concentration the number of embryos produced was double that of the controls. Copper sulphate also reduced the maturation time of somatic embryos to 25 days from embryo initiation. High levels of 2,4-D were detrimental to embryo production. Similarly, fragmented embryos incubated in the dark produced more embryos tan those incubated under light conditions. On the basis of these results, the use of cassava somatic embryo micropropagules for germplasm conservation and synthetic seed development seems to be a strong possibility. Keywords Copper sulphate · Embryogenic calli · Somatic embryo · Dark incubation Communicated by M.R. Davey K.E. Danso · B.V. Ford-Lloyd (✉) School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK e-mail: [email protected]

Introduction Somatic embryogenesis (SE) as an efficient and reproducible regeneration system for the transformation of cassava (Manihot esculenta Crantz) has proven successful in some cultivars (Puonti-Kaerlas 1997; Raemakers et al. 1997). Particle bombardment of embryogenic suspensions of accessions TMS 60444, M. Col 1505 and Bonoua allowed the regeneration of transformed cassava (Taylor et al. 2001). However, the application of SE is not restricted to its transformation capacity alone but can also be used for the rapid multiplication of healthy and disease-free planting materials of cassava. According to Raemakers (1993) the multiplication rate in cyclic SE in cassava is comparatively higher than micropropagation via nodal cuttings. In addition, somatic embryos can be used for long-term germplasm conservation via cryopreservation and synthetic seed development (Zhou et al. 2000). Synthetic seeds promise to be a valuable alternative to stem cuttings of cassava, which are associated with the transmission of viruses and are bulky and highly perishable. However, the conservation of germplasm using somatic embryos is rare. This can be attributed to the low rates of primary embryo induction, the highly genotypic dependent nature of the whole embryogenic system as well as the low rates of plant recovery from somatic embryos. The embryogenic system must be improved to remove these drawbacks if somatic embryos or embryogenic calli are to be used for cryopreservation or synthetic seed development. Several methods, including the pretreatment of donor explants with 2,4-dichlorophenoxyacetic acid (2,4-D) (Raemakers et al. 1997), the addition of paclobutrazol to the culture medium (Li et al. 1995) or the addition of activated charcoal (Matthews et al. 1993), have been used to improve the embryogenic system in cassava. In recent years, the effects of supplementary micronutrients such as Ag+ Co2+, Ni2+, Zn2+and Cu2+ on plant regeneration via SE have received considerable attention (Sahrawat and Chand 1999). In rice (Sahrawat and Chand 1999) and barley (Castillo et al. 1998), the addi-

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tion of copper sulphate to the induction medium increased plant regeneration from immature embryos and embryogenic calli, respectively. In this paper we report on screening cassava accessions for their embryogenic competence on a medium containing 2,4-D and supplementary copper sulphate. The effects of copper sulphate on primary and secondary embryo production as well as light/dark effects on secondary embryo production are also reported.

Materials and methods Plant materials Seeds from ten individual open-pollinated plants of cassava (Manihot esculenta Crantz) accession SM-2075-1 as well as 15 clonal accessions were received from the Centro International de Agricultura Tropical (CIAT) (courtesy of Dr. Fregene). The remaining clonal accessions were obtained from the International Institute of Tropical Agriculture (IITA, Nigeria, 13 accessions) and the Biotechnology and Nuclear Agriculture Research Institute (BNARI, Ghana, five accessions). Clonal accessions were maintained through a 5-week serial subculture on medium containing MS (Murashige and Skoog 1962) basal salts and vitamins supplemented with 30 g l–1 sucrose, 0.05 mg l–1 6-benzylaminopurine (BAP), 0.01 mg l–1 α-naphthaleneacetic acid (NAA), 2 µM CuSO4 solidified with 7 g l–1 agar. The cultures were incubated at a temperature of 24–25°C under a 16/8-h (light/dark) photoperiod with light provided by white fluorescent tubes at an intensity of 40 µmol m–2 s–1.

embryos were defined as having matured when distinct hypocotyls and cotyledons were visible. Additionally, the effect of 4 mg l–1 or 16 mg l–1 2,4-D on somatic embryo production was compared with the effect of 8 mg l–1 using accessions TMS 60444, TME 2019, M. Col 1505, M. Col 1468 and M. Per 183. Effect of copper sulphate on the embryogenic competence of clonal accessions The effect of copper sulphate on the embryogenic competence of clonal accessions was tested on selected accessions based on the results of the screening experiment. Young leaf lobes were cultured on MS basal medium supplemented with 20 g l–1 sucrose, 8 mg l–1 2,4-D and 0, 1, 2 or 5 µM CuSO4. The cultures were incubated as described above. The number of leaf lobes that formed calli and subsequent somatic embryos was recorded. In addition, the number of somatic embryos produced per callus was also counted under the light microscope 14 days after the latter was transferred to the maturation medium. Effect of copper sulphate on cyclic embryogenesis Aliquots (100 mg) of isolated matured somatic embryos from each of TME 2019, TME 279, TME 9 and TME 60444 were fragmented and cultured on induction medium supplemented with 8 mg l–1 2,4-D and 0, 1, 2 or 5 µM copper sulphate. The fragmented embryos were distributed over four petri dishes, each dish containing 25 ml of induction medium, and incubated in the dark. After 14 days, embryogenic clumps were transferred to maturation medium supplemented with 1 µM CuSO4 and incubated in the light. The rates of emergence of matured somatic embryos as well as the number of embryos produced per embryogenic clump were recorded.

Induction of somatic embryos from cotyledons Seeds were soaked in 350-ml honey jars containing 250 ml of tap water overnight to rehydrate. They were then surface-sterilized by immersion in 70% ethanol for 1 min, followed by immersion in 5% commercial bleach (active ingredient: sodium hypochlorite) containing two drops of Tween 20 for 6 min and finally rinsed with three changes of sterilized distilled water. Cotyledons were excised and cultured on MS basal medium supplemented with 20 g l–1 sucrose, 1 µM CuSO4 and 0–16 mg l–1 2,4-D in 8.5-cmdiameter petri dishes containing 25 ml of the induction medium. The medium was adjusted to pH 5.7 prior to the addition of 7 g l–1 agar and autoclaving at 120°C. The growth regulator 2,4-D was filter-sterilized before it was added to the medium. Five cotyledons were placed in one petri dish and incubated in the dark (step 1). After 14 days in culture, calli with or without globular embryos were transferred to the same medium supplemented with 0.1 mg l–1 BAP but without 2,4-D and incubated in light for the maturation of embryos (step 2). Screening for embryogenic competence in clonal cassava accessions Excised young leaf lobes (1–3 mm) from 33 cassava clonal accessions were cultured on MS basal medium supplemented with 8 mg l–1 2,4-D plus 1 µM copper sulphate (step 1) as described above. After 14 days of incubation in the dark, calli with or without embryos were transferred to the maturation medium (step 2), which consisted of MS supplemented with 0.1 mg l–1 BAP, 100 mg l–1 myo-inositol, 1 µM CuSO4, 30 g l–1 sucrose and 1 mg l–1 thiamine HCl, 1.5 mg l–1 pyridoxine HCl, 1.5 mg l–1 nicotinic acid, 2 mg l–1 glycine [the latter four referred to as cassava vitamins (Mathews et al. 1993)]. The number of leaf lobes that formed calli and subsequent embryos were recorded, while the number of somatic embryos that matured was counted under the microscope after 14 days on maturation medium. Somatic

Effect of light/dark on secondary embryo production Aliquots (100 mg) of mature somatic embryos isolated from cotyledons of SM1-2075-1 and SM2-2075-1 were fragmented and recultured on induction medium supplemented with 8 mg l–1 2,4-D for the production of secondary somatic embryos. The cultured fragments were incubated under either light or dark conditions. After 10 days of culture, calli were transferred to maturation medium (S2). The number of mature embryos derived from fragments incubated under either the light or dark conditions was counted after 10 days.

Results Response of cotyledon explants to SE Cotyledons from seeds cultured on hormone-free medium did not form callus or somatic embryos. However, the embryonic axes attached to the cotyledons developed into complete plants with a prominent taproot and shoots after 14 days of culture in the dark. Cotyledons cultured on medium with 2,4-D were swollen within 7 days and developed callus on the adaxial surfaces. Upon transfer of the calli to maturation medium there were varied morphogenic responses: within 10 days, foliose structures, somatic embryos or roots had developed. Calli derived from culture on medium with 4 mg l–l 2,4-D predominantly formed roots with no somatic embryos, while calli that developed in the presence of 8 mg l–l 2,4-D formed somatic embryos.

228 Table 1 Cassava accessions of different geographical origins tested for their embryogenic competence Accessions from Africa

Accessions from Latin America

Accessions

Geographical origin

Classificationa

Accessions

Geographical origin

Classificationa

TME 1808 TME 1801 TME 1939 TME 2019 TME 2037 TME 279 I 4 (2) 1425 TME 60444 TME 2 TME 9 I 30572 TME 3 TME 24 Afisiafi (AFI) Abasa fitaa (ABA) Santom (SM) Tek bankye (TEK) Biafra (BF)

Republic of Benin Republic of Benin Republic of Benin Republic of Benin Republic of Benin Nigeria Nigeria Nigeria Nigeria Nigeria Nigeria Nigeria Nigeria Nigeria Nigeria Ghana Ghana Ghana

Moderate High Very high Moderate Low Very high Low High Non-embryogenic High Moderate Low High Non-embryogenic Low Very high Low Non-embryogenic

M. Col 2215 M. Col 22 CM 523-7 CM 3306-19 CM 3555-6 CM 4365-3 CM 6740-7 M. Col 1468 M. Bra 12 M. Bra 191 M. Bra 383 M. Col 1505 M. Ven 77 M. Per 183 NGA-2

Columbia Columbia Columbia Columbia Columbia Columbia Columbia Brazil Brazil Brazil Brazil Venezuela Venezuela Peru Nigeria

Very high Very high Moderate High Moderate Moderate Moderate Moderate Low Low Low Very high Very high High Moderate

a Very

high: 80–100%; high: 60–80%; moderate: 20–60%; low: less than 20%

Somatic embryogenetic response of young leaf lobes of clonal accessions

Fig. 1 Embryogenic calli from cotyledon of SM1-2075-1 line 1 with somatic embryos. c Callus, se somatic embryos

However, cotyledons from SM3-2075-1, SM4-2075-1 and SM5-2075-1 did not develop somatic embryos on MS basal medium supplemented with 8 mg l–l 2,4-D even after prolonged culture on maturation medium. Cotyledons from SM1-2075-1 and SM2-2075-1 developed somatic embryos (Fig. 1), with more than 50 embryos per cotyledon. Only one cotyledon from SM9-2075-1 developed somatic embryos in the presence of 16 mg l-l 2,4-D, and the number of embryos produced was comparatively lower (fewer than ten).

Young leaf lobes (1–3 mm) from 33 accessions of cassava from BNARI (Ghana), IITA (Nigeria) and CIAT (Columbia) were screened for their embryogenic competence. These accessions represent breeding lines, improved cultivars and landraces of cassava of different geographical origins (Table 1). Leaf-lobe explants formed callus within 7 days of culture on induction medium. The calli were soft, friable or compact and yellowish-cream, cream or brown in colour. Globular or torpedo-shaped embryos were visible on the yellowish-cream or cream embryogenic calli within 14 days of culture, while brown calli seldom developed embryos. Upon transfer to maturation medium, the globular and torpedo-shaped somatic embryos matured within 10 days. In some accessions, embryogenic calli that did not develop somatic embryos developed roots. The percentage of leaf lobes that developed somatic embryos is shown in Fig. 2a, b. Of the 18 accessions from Africa, 15 showed embryogenic competence. The percentage of leaf lobes that produced somatic embryos varied depending on the accession and was not influenced by the geographical origin of the accession (Fig. 2a, b). Of the 18 accessions from Africa, only three did not produce somatic embryos. Among the accessions responding to embryogenesis, TME 2037 showed the least embryogenic competence (8.3%), while TME 279 showed the highest (92.4%). Seven accessions – TME 1801, TME 1939, TME 279, TME 60444, TME 3, TME 24 and Santom (SM) – had more than 60% of their leaf lobes producing somatic embryos, while the remaining accessions had less than 40%. In contrast, all 15 accessions of Latin American origin developed somatic embryos. However, the percent-

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Fig. 3 The effect of 2,4-D on the frequency of embryo production (a) and the mean number of embryos produced per callus (b) Fig. 2a, b The response of leaf lobe-derived calli to somatic embryogenesis on a medium with added copper sulphate. a Accessions from Africa, b accessions from Latin America. Numbers above bars represent the mean number of somatic embryos produced

age of leaf lobes that developed somatic embryos varied depending on the accessions (Table 1). The M. Col series from both Venezuela and Columbia showed very high embryogenic competence, while the CM series from Columbia and M. BrA series from Brazil showed moderate and low embryogenic competence, respectively (Table 1, Fig. 2b). M. BrA 12 had the lowest percentage of leaf lobes (7.7%) developing somatic embryos, while in M. Col 1505 all of the leaf lobes developed somatic embryos. The mean number of somatic embryos produced per responding callus of African origin ranged from 1.0 to 13.5, while in the Latin American accessions it was 1.1–9.5 (Table 1). Based on these results, clonal accessions were classified as being very highly competent (80–100% of embryogenic calli developing somatic embryos), high (60–80%), moderate (20–60%), low (less than 20%) or non-embryogenic (no somatic embryos produced) (Table 1). Effect of 2,4-D concentration on somatic embryo production from leaf lobes We tested the effect of different 2,4-D concentrations (4, 8 or 16 mg l-l) on selected accessions. In contrast to cotyledon explants, young leaf lobes cultured in the presence of 4 mg l-l 2,4-D formed calli, which subsequently developed somatic embryos upon transfer to maturation

medium. However, in all of the accessions tested fewer calli formed somatic embryos on medium with 4 mg l–1 2,4-D than on medium with 8 mg l–1 2,4-D, except for CM 523-7 (Fig. 3). In only two accessions (M. BrA 191 and M. Per 183) was the production of somatic embryos better in the presence of 16 mg l–-1 2,4-D than in the presence of 8 mg l–l2,4-D (Fig. 3). Where 16 mg l–1 was the optimum concentration of 2,4-D, the percentage of calli forming somatic embryos increased with increasing 2,4-D concentration. Among all of the accessions tested, M. Col 1468 had the least embryogenic competence. Percentage embryogenic calli that formed somatic embryos ranged from 12.5 on 4 mg l–1 2,4-D to 42.4 on 8 mg l–1 2,4-D. The number of embryos produced per callus in all of the accessions tested showed a different trend (Fig. 4). It was low on calli derived from culture in the presence of 16 mg l–1, except in M. Per 183. The optimal concentration for embryo production was either 4 mg l–1 or 8 mg l–l depending on the accession. Effect of copper sulphate on somatic embryogenesis Based on the results of the screening experiment using young leaf lobes, clonal accessions were classified as very high, high, moderate, low and non-embryogenically competent with respect to embryogenic competence (Table 1). We selected accessions from each classification group to test for the effect of different concentrations of copper sulphate on embryogenic competence, with an emphasis on accessions that had been classified as having a low embryogenic competence. Embryos in-

230 Table 2 The effect of copper sulphate on secondary embryo production

a Critical

value (P=0.05): 4.20

Concentrationa of CuSO4 (µM)

TME 2019

TME 279

TME 9

TME 60444

0.0 1.0 2.0 5.0

23.75 27.50 40.25 15.25

52.50 58.75 55.50 42.75

53.75 67.00 73.25 55.00

45.25 66.00 68.75 100.75

Table 3 Effect of light/dark incubation of fragmented embryo explants on the number of embryos produced at secondary somatic embryogenesis Genotypea

Incubation treatment

Number of calli clumps cultured

Number of calli with somatic embryos

Total no. of somatic embryos isolated

Mean no. of somatic embryos per callus

SM1-2075-1 (SE line 1)

Dark Light

35 25

34 23

210 60

6.0±0.6 2.4±0.5

SM1-2075-1(SE line 2)

Dark Light

20 20

18 16

119 74

5.9±1.1 3.7±0.7

a SM

1,

Seeds from one particular plant; SE, somatic embryo; line, embryos produced from seed

However, the stimulatory effect of copper sulphate varied from one accession to the other. The optimal concentration of copper sulphate for the induction of somatic embryos was either 2 µM or 5 µM depending on the accession. In TME 2019, Abasa Fitaa (ABA) and Biafra (BF) the optimal concentration was 2 µM; at this concentration, 33.3%, 60% and 90%, respectively, developed somatic embryos. In contrast, for M. Per 183, M. Col 1505, Santom (SM) and Afisiafi (AFI) the optimal concentration of copper sulphate was 5 µM. Accession TME 2 had been classified as being non-embryogenic, but in the presence of 2 µM copper sulphate 3.7% of the calli developed somatic embryos. Similarly, the mean number of embryos produced was also comparatively higher in the treated calli than in the controls in all of the accessions (Fig. 4). In Biafra and M.Col 1505, the optimal concentration was 2 µM, while in the remaining accessions tested, 5 µM was optimal. The highest number of embryos produced per callus (12) was obtained in M. Col 1505. The effects of copper sulphate on secondary embryo production Fig. 4 The effect of copper sulphate on the frequency of embryo production (a) and the mean number of embryos produced per callus (b)

duced on media supplemented with copper sulphate had well-developed green cotyledons within 14 days, indicating early maturity, compared to the controls where most of the embryos were in the late-torpedo stage or earlycotyledonary stages. The number of embryogenic calli that subsequently developed somatic embryos was also comparatively higher on the copper sulphate-supplemented medium (Fig. 4).

Fragmented somatic embryos cultured on maturation medium containing supplementary copper sulphate produced embryos within 7 days of transfer to maturation medium. The effect of copper sulphate on the production of secondary somatic embryos is shown in Table 2. The presence of supplementary copper sulphate in the induction medium significantly (P=0.05) increased somatic embryo production over the controls, except at the highest concentration of copper sulphate where there was only a significant increase in the number of embryos in TME 60444. In three of the accessions there were no significant differences between 1 µM and 2 µM copper

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sulphate, indicating that the optimal concentration of copper sulphate was highly dependent on the genotype. In TME 2019 and TME 9, 2 µM was optimal. There was a significant (P=0.05) positive interaction between copper sulphate and the accessions when a two-way analysis of variance was performed. The effect of light/dark incubation on the production of secondary somatic embryogenesis Primary embryos from SM1-2075-1 and SM2-2075-1 were used to study the effect of light/dark incubation on the number of embryos produced. Somatic embryos produced under a dark/light incubation regime developed mature somatic embryos (within 7 days) earlier than those cultured under continuous light. Fragmented primary embryos of SM1-2075-1 incubated in the dark followed by light incubation at the maturation stage produced more embryos than explants incubated under continuous light at the induction and maturation stages (Table 3).

Discussion In cassava, cotyledons and young leaf lobes are the two main explants used for the induction of primary somatic embryos, with cotyledons being more embryogenically competent than leaf lobes. We observed a high frequency of embryo production when we cultured these explants on a medium supplemented with 8 mg l–1 2,4-D plus 1 µM copper sulphate. However, due to high heterozygosity and poor seed production in some cultivars, leaf-lobe explants are frequently used for somatic embryo production. Of the 33 clonal cassava accessions we tested for embryogenic competence, 30 produced somatic embryos. An increased concentration of copper sulphate in the medium increased the percentage of leaf-lobe explants that developed somatic embryos and also doubled the number of embryos produced per callus relative to the controls. The high response could be due to the addition of supplementary copper sulphate to the induction medium. Using the same protocol without supplementary copper sulphate, Szabados et al. (1987) reported that 60–70% of leaf lobes of cassava accession M. Col 1505 formed somatic embryos. Mathews et al. (1993) incorporated copper sulphate into the induction medium but did not add BAP to the maturation medium, and 85% of the leaf lobes developed embryos. In our study, 100% of the leaf lobes of M. Col 1505 developed somatic embryos. Raemakers (1993) did not obtain any somatic embryos from M. Ven 77 on a medium supplemented with 8 mg l–1 2,4-D, while in our study 80% of the cultured explants of M. Ven 77 produced somatic embryos when cultured with copper sulphate. Copper sulphate is known to improve plant regeneration from immature embryos in rice (Sahrawat and Chand 1999) and embryogenic calli in barley (Castillo et

al. 1998). While the exact function of copper sulphate in plant regeneration has not been determined, it is known that copper sulphate is a component or activator of many enzymes involved in electron transport, photosynthesis and protein and carbohydrate metabolism. The stimulatory effect of copper sulphate could be important by playing a key role in morphogenesis at an optimum concentration (Sahrawat and Chand 1999). It is also known that copper sulphate at concentrations of 1.0–25.0 µM inhibits ethylene, a modulator of plant growth in a hydroponic rice nutrient solution (Lidon et al. 1995). Copper sulphate may therefore enhance somatic embryo production through its inhibitory effect on ethylene. El Meskaoui and Temblay (2001) demonstrated that limiting ethylene biosynthesis by the addition of inhibitors to the maturation medium of low-capacity embryogenic lines of black spruce promoted somatic embryo production. The incubation of fragmented embryos in darkness followed by a transfer to light produced more somatic embryos within a relatively short time than did incubation under continuous light. Some species, such as poplar and Podophyllum, have an absolute requirement for dark during embryogenesis (Merkle et al. 1995). However, cassava fragmented embryos require an initial dark incubation period before the transfer to maturation medium for embryo maturation. Successful transformation procedures in cassava via particle bombardment (Schöpke 1996; Raemakers et al. 1996; Taylor et al. 2001) or Agrobacterium mediation (Gonzalez et al. 1998; Schreuder et al. 2001) rely on SE for both gene insertion and recovery of transgenic plants. Thus, it is essential that cassava accessions, independent of their geographical background, are made embryogenically competent to enable transformation procedures to address agronomic problems such as susceptibility to cassava mosaic virus diseases. Of the 30 responding accessions, there were no differences in embryogenicity, indicating a strong possibility for genetic transformation independent of their origin. Our results compare with those of Taylor et al. (2001) who produced friable embryogenic callus of a similar nature in 14 cultivars of different geographical origin; eight of these have successfully been regenerated into plants, and transgenic plants have been obtained from three. The application of SE in micropropagation and germplasm storage requires an efficient system of embryo production. In our study we have shown that the addition of copper sulphate enhanced early maturation of the somatic embryos; somatic embryos matured within 25 days compared to the 35 days reported by Matthews et al. (1993). We have also shown that incubation of fragmented embryos in the dark produced more somatic embryos within a relatively short time than did incubation under continuous light. Furthermore, we have demonstrated that an increased concentration of copper sulphate in the induction medium increased embryogenicity in all accessions as well as doubled the number of embryos produced. Thus, a system for the rapid generation of high-frequency so-

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matic embryos independent of accessions (genotype) has been obtained, making somatic embryos alternative micropropagules for the long-term conservation of plant genetic resources and, more importantly, a potential means of propagation on an agricultural scale. Somatic embryogenesis has the potential to accelerate the introduction of improved cultivars into commercial production, since they can be encapsulated and handled as seeds. However, further investigations on the frequency of conversion of somatic embryos into plants and an optimization of cryogenic protocols are needed to make somatic embryos of cassava more useful as ideal micropropagules for both the mass multiplication and long-term conservation of cassava. Acknowledgements The authors wish to thank The Commonwealth Scholarship Commission, UK for their financial support to Mr. Kenneth Ellis Danso. We also thank Mrs. Ng at IITA and CIAT for sending us in vitro plantlets of cassava for this investigation.

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