Primary Somatic Embryos from Axillary Meristems and Immature Leaf Lobes of Selected African Cassava Varieties

The study evaluated high-value African cassava varieties for primary somatic embryogenesis using axillary meristems (AM) and immature leaf lobes (LL) on piclorambased medium. The study was conducted at the Central Biotech Lab, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria between 2006 and 2009. Completely randomized design with four replicates was used for the study. Using LL explants, there were significant (P=.05) differences in percent responding leaf lobes, percent explant with pre-embryogenic structure, PSEF and PSEE among cassava varieties. The PSEF of the only three varieties that produced mature somatic embryo were 93.6, 88.5 and 85.7% for TME 12, Kibaha and Albert, respectively. Similarly, significant (P=.05) differences existed among the varieties in percent enlarged axillary meristem, percent explant with pre-embryogenic structure, PSEF and PSEE when AM Research Article British Biotechnology Journal, 3(3): 263-273, 2013 264 was the explant. The PSEFs of the only three varieties that produced mature somatic embryo were 83.6, 77.5 and 72.7% for TME 12, Kibaha and Albert, respectively. The PSEF and PSEE of LL explant were greater than those of AM by an average of 86.1% and 82.7%, respectively. The study concluded that both AM and LL were good explants for production of primary somatic embryo in cassava.


INTRODUCTION
Cassava is an important source of energy in the diet of livestock and about 600 million people in tropical and subtropical climates [1].The increase in cassava cultivation and its important role in food security are enhanced by two factors: the unique biology of the crop and the numerous uses to which its starch and by-products are put [2,3,4].The starch content of cassava roots ranges from 65 -91% of its total root dry weight depending on the cultivar [5,6].The global demand for cassava starch is rising and it is fastly replacing conventional sources of starch like wheat, maize, rice and potato because of its enhanced properties [7,8].Despite its potentials for achieving food security and economic growth, biotic and abiotic constraints such as diseases, pests, weeds, poor soil fertility and drought are militating against cassava production [9,10,11].Application of conventional breeding methods for improvement of cassava against the biotic and abiotic constraints has so far recorded limited success [9,12].Conventional breeding of cassava is challenging due to the highly heterozygous nature of the crop which prevents a backcross scheme.In the field, cassava is typically propagated clonally by stem cuttings.This propagation strategy is ideal for a bio-engineering approach to crop improvement as gene segregation throughout crossing is limited [13].Successful genetic modification by bio-engineering approach requires establishment of in vitro regeneration and transformation systems.To date, the only reported means of incorporating foreign pieces of DNA into cassava genome is via regeneration through somatic embryogenesis [13].
Plant regeneration via somatic embryogenesis has become an integral component of genetic transformation system in cassava [14,15].Regeneration studies have shown that the frequency and efficiency of somatic embryogenesis are highly genotype-dependent, and not all cassava cultivars are amenable to somatic embryogenesis, regeneration and transformation, therefore, there is a need to optimise the generation of embryogenic structures for each cassava cultivar [16,17,18,19,20,21,22].As a result, more than 60 African cassava cultivars and other cultivars from South America and Asia have been tested for their somatic embryo-producing ability.For the same reasons, somatic embryos have been induced from various cassava explants including immature leaf lobes [16,17,18,19,20,21,22,23,24], shoot apical meristems [16,20,24], zygotic embryos or floral tissue [23], on several media containing various plant growth regulators.
Induction and maturation of somatic embryo from African cassava cultivars have been mostly obtained from immature leaf lobes and shoot apical meristem explants [16,20].Limited number of studies reported the use of axillary meristem as explants.Rossin and Rey [24] obtained primary somatic embryo from axillary meristem of eight South African cassava cultivars with primary somatic embryo frequencies and efficiencies ranged from 28 to 83% and 1.5 to 4.0, respectively.Compared with apical meristem or leaf lobe explants, axillary meristems occur in large number on plantlets with the advantage of scaling up experiment as high number of axillary meristem can be obtained from in vitro plantlets.High number of explants per replicate or experiment increases the chance of obtaining positive response to somatic embryo induction, regeneration and transformation.In addition, the use of axillary meristem provides opportunity to increase the scope of research by including several factors for investigation which expands the information obtainable from such study.In this communication, eleven African cultivars of diverse agronomical traits were screened for somatic embryo using axillary meristems and immature leaf lobes as explants.

Plant Materials
Cassava (Manihot esculenta) plantlets of eleven cultivars (Table 1) were obtained from in vitro germplasm collection of International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria.The plantlets were maintained by regular subculturing at four weeks interval as in vitro shoot cultures on basal medium (BM).

Basal Medium and Culture Conditions
Basal medium (BM) which, consisted of full-strength MS [25] salt (Sigma, USA) along with 0.8% (w/v) agar (Oxoid Ltd, England), 30 g/l sucrose and 2µM CuSO 4 , was used in all experiments unless otherwise stated.The pH of the medium was adjusted to 5.8 by HCl (1 N) or NaOH (1N) prior to autoclaving at 121ºC for 15 minutes at 1.05 kg cm -2 pressure.Growth regulators were filter sterilized through 0.22-μΜ Millipore filters and added to media after autoclaving.For all experiments, cultures were maintained under 16 h photoperiod with 20 µmol m -2 s -1 light intensity provided by cool-white fluorescent tubes at 25 ± 2ºC.

Induction and Maturation of Primary Somatic Embryos from Axillary Meristems
The procedure as described by Rossin and Rey [24] was followed for induction of primary somatic embryo from axillary meristem isolated from axillary bud.Nodal explants (2-3 cm) were excised from three-week old in vitro plantlets and incubated on BM containing 10 mg/l BAP in the dark for 7 days for enlargement of axillary buds.Lateral meristems (1-2mm) were isolated from the enlarged axillary buds with sterile surgical blade and forcep with the aid of microscope in a horizontal laminar flow bench.The isolated axillary meristems were incubated in BM supplemented with 10 mg/l of picloram for 17 days for induction of primary somatic embryo.Pre-embryogenic structures were later incubated in dark on BM supplemented with 0.1mg/l BAP for 10 days for maturation.

Induction and Maturation of Primary Somatic Embryo from Immature Leaf Lobes
Primary somatic embryo was induced from immature leaf lobe as previously described by [20].Immature leaf lobes (1-6 mm) obtained from three-week old in vitro plantlets were incubated in the dark on BM supplemented with 10 mg/l picloram for 14 days.Preembryogenic structures were later incubated in the dark on BM supplemented with 0.1mg/l BAP for 10 days for maturation.

Experimental Design and Statistical Analysis
All experiments were arranged in completely randomized design with four replicates in each experiment.Each experiment was repeated three times.Primary somatic embryogenesis frequency was defined as the number of explants that produced somatic embryo and expressed as a percentage.Primary somatic embryogenesis efficiency was the number of somatic embryo produced by an explant and expressed as number of somatic embryo per explant.Observation on number of explants with enlarged meristem was made seven days after incubation.Data on the presence of pre-embryogenic structure and matured embryo were collected on 14 and 21 days after incubation, respectively.Data were subjected to arcsine and square root transformations to normalize variances.Data were further subjected to analysis of variance to detect differences among treatments at P≤0.05 by Duncan's multiple tests as outlined by [26].

Induction and Maturation of Primary Somatic Embryo from Axillary Meristems
Induction and formation of primary somatic embryo via axillary meristem are shown in Fig. 1.Stem segments, each with an axillary node (A), were obtained from three-week old in vitro propagated plantlet.Buds break and elongation were found on the axillary node when cultured for 7 days in the dark on BM containing 10mg/l BAP.The auxillary node on A was successfully enlarged with BAP after seven days of dark incubation.B was an isolated meristem from the enlarged node in A which was used to generate embryogenic callus (C) using picloram.Maturation of C with little quantity of BAP produced D with globular primary somatic embryo which fully matured two weeks later as shown in E. All varieties had enlarged node when cultured on meristem enlargement medium however, significant difference (P=.05) existed in the percentage of enlarged nodes among varieties (Table 2).Cassava varieties Albert, TME 12, Kibaha, TMS 4 (2) 1425 and TMS 98/0505 recorded not less than 70% explant with enlarged node.Only four varieties produced pre-embryogenic structure when transferred on induction medium.There was significant difference (P=.05) in percentage of explants with pre-embryogenic structure among varieties (Table 2).TME 12 recorded the highest (84.6%) percentage of explants with pre-embryogenic structure while TMS 98/0505 had the lowest (10.3%).Pre-embryogenic structure matured into primary somatic embryo in only three varieties which were Albert, Kibaha and TME 12. Variety TME 12 had the largest percentage of mature primary somatic embryo (83.4%)while Albert recorded the lowest (72.7%).The average number of primary somatic embryo per explant in Kibaha (5.6) was significantly higher (P=.05) than Albert (4.5) and TME 12 (4.5).Primary somatic embryos were mostly emerged at the base of explant in Albert, while they emerged from the top of the explant in TME 12 (Table 2).In Kibaha, primary somatic embryos were evenly distributed on explant.Primary somatic embryos produced by Albert were mostly separated in shape while that of Kibaha and TME 12 had fused and conical cotyledon, respectively.

Induction and Maturation of Primary Somatic Embryo from Immature Leaf Lobes
Induction and maturation of primary somatic embryo from immature leaf lobe in TME 12 is shown in Fig. 2.An immature leaf lobe obtained from two-week old in vitro propagated plantlet was decapitated (Fig. 2A).Decapitated leaf lobe inducted for primary somatic embryo with picloram produced Fig. 2B after one week of dark incubation, embryogenic callus after two weeks (Fig. 2C) of dark incubation.Maturation of the embryogenic callus was done with BAP gave D and fully matured primary somatic embryo (E) was successfully obtained after five weeks of dark incubation.The green colour of decapitated immature leaves disappeared as calluses were developing.Callus started developing at 4 days after incubation from the cut edge of the leave and extended into the interior in the responded leaves.Pre-embryogenic structures occupied the central portion of the calluses.The preembryogenic structures are globular in shape with bipolar structures and light-brown in colour (Fig. 2D).There were significant differences (P=.05) in percentages of responded leaf lobe, percent explants with pre-embryogenic structures, frequency and efficiency of primary somatic embryo (Table 3).Variety TME 12 recorded the highest percentage of responded leaf lobe (87.5%) while TMS 1425 had the lowest (62.5%).Only Albert, Kibaha and TME 12 produced pre-embryogenic structures.The percentage of pre-embryogenic structures was highest (84.6%) in TME 12 and lowest (77.4%) in Kibaha.Only three varieties produced mature primary somatic embryo.They were TME 12, Kibaha and Albert with 93.6%, 88.5% and 85.7%, respectively.The efficiency of primary somatic embryo was highest in Kibaha (5.6 embyo/explant).Primary somatic embryos were located at the base of Albert explants, top of TME 12 explants and evenly distributed on the explants of Kibaha.The shapes of primary somatic embryos in the three varieties were: conical in Albert, fused cotyledon in Kibaha and separated cotyledon in TME 12.  Comparing somatic embryo production of immature leaf lobe explant with axillary meristem explant in the three landraces that produced mature primary somatic embryo, primary somatic embryogenesis frequencies and efficiencies in immature leaf explant were greater than in axillary meristem explant by an average of 86.1% and 82.7%, respectively.

DISCUSSION
Production of somatic embryo is critical for development of transgenic cassava plants because somatic embryo is the often used target tissue for insertion of foreign genes [13].In this study, eleven varieties were screened for induction and maturation of primary somatic embryo using immature leaf lobe and axillary meristem.Out of the 11 varieties screened for primary somatic embryogenesis using LL explant, only three varieties (Albert, Kibaha and TME 12) produced mature somatic embryo, which are landraces.Previously, only three varieties (TMS 4 (2) 1425, TME 12 and TMS 91/02324) have been screened for somatic embryogenesis using LL and APM explants (Table 1).Out of the three varieties, mature primary somatic embryos were obtained in TME 12 and TMS 91/02324 varieties from both LL and shoot apical meristem explants (Hankoua et al., 2005).TMS 4 (2) 1425 formed only pre-embryogenic structures and failed to develop to mature embryo.The PSEF and PSEE observed in the present study are comparable with previous reports.A range of 88.5 to 93.6% and 4.5 to 5.6 embryos per explant were observed in this study as PSEF and PSEE, respectively in the three varieties that produced primary somatic embryo.Hankoua et al. [20] reported a range of 48-100% in PSE frequency for a wide range of cassava varieties.Specifically, PSEF (93.6%) and PSEE (4.5 embryo per explant) for TME 12 using LL explants observed in the present study were comparable to PSEF (94.0%) and PSEE (3.7 embryos per explant) reported by Hankoua et al (2005).Recently, a range of primary somatic embryogenesis frequencies of 4 to 89% was reported using LL as explant on picloram-based medium in eight cultivars of South African origin by [24].For the same cultivars, the efficiency of primary somatic embryogenesis ranged between 1.5 and 4.0 using LL as explants.
This study is the first documented attempt at producing primary somatic embryo from axillary meristem in cassava in a wide range of cassava varieties.Though limited variation existed in enlargement of node among cassava varieties, formation of pre-embryogenic structure was largely under genetic influence.Similar reports were made on formation of pre-embryogenic structure from immature leaf lobe in cassava [20,21].The successful enlargement of node in culture media does not translate to induction of pre-embryogenic structure.However, there appear to be a strong relationship between formation of pre-embryogenic structure and maturation of primary somatic embryo as 75% of varieties that formed pre-embryogenic structure produced mature primary somatic embryo.In the present work, the PSEF ranged from 72.7% to 93.6%.Using shoot apical meristem as explant, the frequency of primary somatic embryogenesis were 77 and 93% for TME 12 and TMS 91/02324, respectively as reported by [20].Feitosa et al. [27] reported 15.5 -80.0%frequency of primary somatic embryo and 18.9-24.4embryo per explant in some cassava genotypes of Brazil, using shoot tip explant on picloram-supplemented medium.The successful production of primary somatic embryo using axillary meristem as explant in some cassava varieties established the fact that somatic embryo can be obtained from lateral meristem, apart from apical meristem which had earlier been reported by [20,28,29,30].Mature primary somatic embryo were produced in landraces (Albert, Kibaha and TME 12) while pre-embryogenic structure was obtained only in one improved variety (TMS 98/0505), which did not develop to mature embryo.This result suggested that picloram-based induction medium may be suitable for cassava landraces.Recalcitrance of improved cassava varieties to induction and maturation of primary somatic embryo has been reported [20].The result suggested that variety influenced the location of the embryo on explant and shape of the embryo.

CONCLUSION
The greater values of PSEF and PSEE of LL explant than AM explant suggest that the use of immature leaf lobe explant has potential for production of a large number of explants for secondary embryogenesis, regeneration and transformation studies.

Table 1 . Source, agronomic trait and adapted agro-ecologies of some landraces and improved cassava varieties included in the study
Values are means (±standard error).Values followed by different superscripts in same column are significantly different at P≤0.05 by Duncan's multiple range test.CV= coefficient of variation C=conical cotyledon FC=fused cotyledon SC-separated cotyledon