Efficient plant regeneration protocol for finger millet [Eleusine coracana (L.) Gaertn.] via somatic embryogenesis

1 Institute of Biotechnology Research, Jomo Kenyatta University of Agriculture Technology, P. O. Box 62000-00200, Nairobi, Kenya. 2 Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844-00100, Nairobi, Kenya. 3 South Eastern Kenya University, P. O. Box 170-90200, Kitui, Kenya. 4 International Crops Research Institute for the Semi-Arid-Tropics, P. O. Box 39063, Nairobi, Kenya. 5 Department of Biochemistry and Biotechnology, Pwani University, P. O. Box 195-80108, Kilifi, Kenya.


INTRODUCTION
Finger millet [Eleusine coracana (L.) Gaertn.], which is a small cereal crop that is indigenous to East Africa, is cultivated in arid and semi-arid areas of eastern Africa and south Asia (Sood et al., 2016). It is tetraploid (2n = 4x grain crops for food and nutritional security, climate resilient farming and agricultural diversification. Despite its significance as a subsistence crop, little attention has focused on the crop's improvement programs, probably since it is considered with minimal monetary significance in contrast to maize, wheat and rice. Finger millet production and yield is constrained by lack of improved varieties, weeds, diseases and pests, limited uses, unpredictable markets, limited research and moisture stress in dry areas (Oduori, 2005). Varieties with new traits emerging as one will help the plant cope with adverse challenges associated with biotic and abiotic stress. The world's increasing demand for finger millet offers a golden opportunity to develop efficient, quick and reproducible strategies and techniques for important finger millet local varieties addressing complex traits such as grain quality, biotic and abiotic stress resiliencies. Over the years, conventional breeding has been used for traits improvement in finger millet with some success. However, conventional plant breeding is tedious, time consuming and mostly dependent on environment (Miah et al., 2013). As an alternative, biotechnological techniques such as genetic engineering and genome editing techniques, which relies on the availability of in vitro plant regeneration systems, provides a powerful tool for genetic manipulation of finger millet. Genetic engineering of finger millet for improved varieties is stymied because of absence of a proficient plant tissue culture strategy with high regeneration frequency. To the authors' knowledge, there is no report on tissue culture and transformation for African finger millet cultivars. Just like other cereals, finger millet regeneration can be accomplished through somatic embryogenesis and organogenesis. Somatic embryogenesis of finger millet is one of the most preferred approach due to production of large numbers of plantlets and its application for genetic transformation technology. Several factors such as the explant source, their developmental stage and plant growth regulators affect this morphogenetic route (Sudhakar et al., 2004). Finger millet has been considered a recalcitrant crop to tissue culture and genetic transformation. Only limited reports are available to date on regeneration and genetic transformation of finger millet finger millet varieties particularly of African origin (Gupta et al., 2017). In these reports, the low rate of embryo initiation, maturation, germination and development into plantlets often remain a major challenge. The present work reports a robust and reliable procedure for the establishment of an efficient and reproducible regeneration through system somatic embryogenesis of finger millet using shoot tips that will be useful for the genetic improvement of this crop. Ngetich et al. 661

Plant material and explant preparation
Seeds of finger millet: GBK-043137, GBK-043128, GBK-043124, GBK-043122, GBK-043094 and GBK-043050 were obtained from Kenya Agricultural and Livestock Research Organization gene bank, at Muguga, Kenya. The seeds were soaked for 30 min in sterile distilled water to dehusk them, followed by surface sterilization with 70% ethanol and 20% sodium hypochlorite containing a few drops of Tween 20 for 20 min. Surface sterilized seed were rinsed three times with sterile distilled water and germinated aseptically on Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962), supplemented with 3% sucrose, 0.3% gelrite and pH 5.8. The culture bottles were incubated at 25±2°C in the dark for germination for three days.

Callus induction and somatic embryo development
Shoot tips (4 to 6 mm) from in vitro germinated plants were aseptically excised and cultured on callus induction medium (CIM) for six weeks in the dark. The CIM comprised of MS supplemented with 30% sucrose and various plant growth hormones (PGRs) either singly or in combinations comprising of NAA (2.0, 2.5, 3.0 and 4.0 mg/l); 2,4-D + BAP (1.5 + 0.5, 2.0 + 1.0, 2.5 + 1.5, 3.0 + 1.5 mg/l); 2,4-D + KN (1.5 + 0.5, 2.0 + 0.5, 2.5 + 1.0, 3.0 + 1.0 mg/l) and NAA + KN (1.5 + 0.5, 2.0 + 0.5, 2.5 + 1.0, 3.0 + 1.0 mg/l). Explants cultured on MS media without plant PGRs were taken as control. Cultures were incubated at 25±2°C in the dark for about four weeks to promote the formation of callus. The explants were subcultured to a fresh media after every two weeks. Calli that formed were transferred onto embryo induction medium (EIM) containing MS basal salts supplemented with 30 g/l sucrose and 1.75 mg/l of BAP and incubated at 25±2°C in dark for 8 weeks until differentiation into embryo-like structures were observed. The calli were subcultured onto fresh EIM medium after every two weeks.

Shoot proliferation and root formation
Mature embryos were cultured on shoot induction medium (SIM) comprising of MS supplemented with 30% sucrose and different concentrations of BAP (1, 1.5, 1.75 and 2 mg/L). The SIM was also used for elongation with subculture every two weeks up to four subcultures. The number of shoots formed were counted every four weeks and recorded. Shoots formed were cultured on root induction media (RIM) comprising of MS medium supplemented with 30% sucrose and 1.0 KN+0.25 NAA, 1.0 BAP + 0.25 NAA, 1.0 KN + 0.25 2,4-D and 1.0 BAP + 0.25 2,4-D for root formation and development for four weeks. The developed roots were observed and recorded.

Hardening and acclimatization
Rooted plantlets were rinsed with sterile distilled water to remove the excess media. The plantlets were transferred to the greenhouse for acclimatization on four media regimes: forest soil only, cocopeat, forest soil + sand + manure (2:1:1) and forest soil + sand + fertilizer (4:2:0.05). The plants were watered on regular intervals. Data on *Corresponding author. E-mail: w.mbinda@pu.ac.ke.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License and on the sixth week from the day of hardening off. Survival of plantlets was recorded after 3 weeks [Survival plantlet (%) = (surviving plantlets/total plantlets) x 100).

Statistical analysis
All data were expressed as mean ± standard. The data were analysed using ANOVA with Minitab statistical computer software v.17. Means were separated using the Fisher's protected LSD test at a confidence level of 95% (p ≤ 0.05).
The callus induction mean percentage ranged from 4.33±0.33 to 12.33±0.33 out of 60 explants based on the type and concentration of PGRs in the medium ( Table 2). The highest callus formation was observed on medium supplemented with 2.5 mg/l 2,4-D + 1.5 mg/l BAP at 12.33±0.33 for variety GBK-043128 (Table 2a). This combination and concentration also had the best response for callus induction on all other varieties when compared with the others. GBK-043094 was the best responding variety with regards to callus induction in all media combinations and concentrations. Medium supplemented with NAA was the worst performing media with 4.33±0.33 callus formation. This medium formed callus like structures at the tip of explant and developed shoots within three days which were fast growing. These explants were subcultured; they turned brown after a week and eventually died off. The controls did not form any callus (Table 2a and b). Explants formed calli that were friable, soft, watery, white in colour, they developed gradually, slowly and were non-regenerative with unorganized morphology, destitute of nodular structures (Figure 1b). Following a month of culture, the greater part of the calli turned out to be light yellow in colour and embryogenic in nature with nodular development ( Figure  1c). Explants did not show any change turned brown and eventually black and they died within three weeks of culture.

Regeneration of plants from callus
The rapidly growing friable calluses were subcultured onto MS medium supplemented with 1.75 mg/l of BAP for somatic embryo induction. Somatic embryogenic structures started to form after two weeks culture onto the EIM. The greenish compact and moderate developing calli that developed organized structures and inevitably displayed tissue differentiation were regarded as embryogenic (Figure 1d), as opposed to the nonembryogenic calli that were white or cream, friable and quickly developing (Figure 1e). Some calli of the finger millet varieties turned green and formed globular, heart, torpedo and cotyledonary stages of embryos which appeared progressively after 4 weeks on EIM medium. There was 100% transition from callus to somatic embryos for all finger millet varieties. After four weeks on EIM in light, the embryo maturation was observed with formation of small shoots (Figure 1f).

Shoot elongation and multiplication
The highest shoot formation frequency was achieved using MS supplemented with 1.75 mg/l on GBK-043094 and GBK-043137 both with a mean of 25.07±0.64 and 25.33±2.95, respectively (Table 3). This concentration of MS and 1.75 mg/l was also highly significant (P≤0.05) for GBK-043128, GBK-043124, GBK-043050, GBK-043094 and GBK-043137 with mean number of shoots at 23.93±0.74, 22.53±1.03, 23.93±0.74, 25.07±0.64 and 25.33±2.95 respectively, however a lower concentration of 1.5 mg/l was highly significant for GBK-043122. The lowest performing shooting media was 1.0 mg/l on GBK-043050 and 2.0 mg/l on GBK-043050 with a mean number of shoots of 9.80±1.33 and 9.80±2.40 respectively. The height of plants varied from 3 to 6 cm.

Root induction
Shoots (2 to 3 cm in height) cultured on MS media    Table 4). The best rooting hormone combinations not only had highest rooting but also 100% of the plants formed roots as compared to rest of the combinations.

Hardening and acclimatization
Well-developed plantlets (5 to 6 cm in height) with more than two true leaves from all the regenerated varieties were hardened successfully in pots using four media regimes: forest soil only, cocopeat, forest soil + sand + manure in a ratio and forest soil + sand + fertilizer in 2:1:1 and 4:2:0.05 ratios with 97% survival rate (Figure 1h to i). The number of leaves for all the in vitro regenerated finger millet varieties varied between 6.43±1.30 and 11.92±0.96. The number of leaves for all the in vitro regenerated finger millet varieties was statistically, not significantly different at two and four six weeks for soil while all the rest were different (Table 5) across the media regimes. Following seven days of incubation in a growth chamber, all the pots were moved to regular environmental conditions with a 100% survival rate with a 100% survival rate. The somatic embryo-derived plantlets grew well and exhibited phenotypic homogeneity as compared to seed-derived field-grown finger millet plants (Figure 1g and h). The height of the plants at two weeks varied from 3 to 6 cm. The shortest plants at this period were those hardened on soil, sand and manure which were also significantly different on GBK-043124 (Table 6). At four weeks, the tallest plants were produced using cocopeat which was not significantly different across varieties (Figure 1b). The media with the tallest plants at six weeks was soil, sand and manure (52.33±5.56) on variety GBK-043128 which was significantly different while soil + sand + fertilizer produced the shortest plants (Table 6).

DISCUSSION
In this study, shoot apical meristems derived from mature seeds of several finger millet varieties germinated in culture used for the successful induction of embryogenic calli and subsequent plant regeneration. Although, six Kenyan farmers preferred finger millet varieties were used, this regeneration system can be extended into a range of agronomically important African finger millet varieties. Explants derived from mature seeds are considered an excellent source material for biotechnological application due to easy storage and accessibility to large amounts of uniform quality explant material (Sudhakar et al., 2004). Shoot apical meristems have been used successfully in the regeneration systems in cereals as starting material to obtain stable transformation in barley, wheat, maize, sorghum and millet (Sticklena and Orabya, 2005). The use of shoot apex in regeneration is critical since it can divide to produce viable new organs such as leaves, stems and adventitious roots (Itoh et al., 2006). Shoot apex is also most beneficial for its quick development therefore it can allow rapid development of plants (Ceasar and Ignacimuthu, 2008;Dey et al., 2012). Auxins have been shown to play an important role in inducing callus (Anjaneyulu et al., 2011). Therefore, in the current study, it was used alone or in combination with cytokinins. It was observed that by increasing levels of NAA across varieties resulted in the formation of callus-like structures at the base of the tip of explant then it started elongating and eventually shoots were formed which was higher as compared to other plant growth regulators. This trend is consistent with the works of Ceasar and Ignacimuthu (2008) who observed that after 5 weeks, NAA induced callus remained non-responsive. The previous reports on cereals tissue culture dealt with different auxins and cytokinins at different concentrations also showed the superiority of 2,4-D over the other auxins. The presence of NAA growth regulator was also found to be inhibitory for plant regeneration as well as callus proliferation. Medium supplemented with NAA was the worst performing media with 4.33±0.33 to 10.00±0.58 callus formation (Table 2a). It is therefore not understood why NAA performed poorly on the finger millet varieties tested. More studies should be done to investigate this callus proliferation as well as plant regeneration inhibitory performance.
Plant growth regulators auxins alone and cytokinins or in combination plays a very important role inducing callus and its proliferation (Thomas and Maseena, 2006). The degree of embryonic callus was improved by a combination of 2,4-D and BAP at 2.5 mg/l 2,4-D +0.5 mg/l of BAP for GBK-043137, GBK-043128, GBK-043122 and GBK-043050. A higher level of 3.0 mg/L 2,4-D + 1.5 mg/L was significant for GBK-043124 and GBK-043094. This is in agreement with the works of Anjaneyulu et al. (2011) who used 2 mg/l ,4-D + 1.0 mg/l of BAP obtaining Table 5. Mean number of leaves at two, four and six weeks hardened using soil, cocopeat, soil, sand and manure, and soil, sand and fertilizer at the greenhouse.