Surface sterilization and rate of initiation
Surface sterilization with ethanol alone has not controlled the endophytic contaminants; hence 0.1% of mercuric chloride tried later has led to 80% explants free of contamination. Among the contamination free explants 90% have initiated an average of 2.5 shoot in MS initiation medium with 2 mg BAP and 1 mg/l Kinetin. Contamination free plantlets transferred for multiplication to fresh media performed well with a record mean number of plantlets 8.5 per explant (Table 1 and Fig. 2C) High loss of plantlets due to contamination was minimized to 10% on average by clean lines control and subsequent sub-culturing. Previous works reported also the contamination rate at early initiation stage is very high due to endogenous latent contaminating microbes (Ayenew et al 2012; Berihu 2018).
The ANOVA result for the in vitro regeneration variables among the 14 treatments showed that there is a highly significant difference among treatment means for all six measurements. Three treatment combinations; 1, 5 and 10 (Table 1) to NH4NO3 replacement were observed as promising sources of nitrogen as compared to standard MS media (13th treatment). MS medium with omitted NH4NO3 and replaced by 1 g/l NH4Cl resulted in a non-significant number of shoots and length with standard MS media containing 1.65 g/l NH4NO3. Average shoots in number (9.33) for NH4Cl supplied media and 10.00 shoots per explant were recorded for standard MS media indicating there is no significant difference between the two-treatment means (Table 1). MS medium supplemented with 3.8 g/l KNO3 to compensate for N2 from ammonium nitrate showed the second highest mean number of shoots (7.33) per explant. For the third alternative tested urea, which is the common inorganic nitrogen fertilizer medium containing 3 and 4.5 g/l produced 7.0 mean numbers of shoots per explant. Another in vitro performance parameter recorded is the number of active buds on micro rhizomes under shoot clumps which can grow to new shoots in next sub-culturing. The highest mean number (7.33) for this was also recorded for the medium of 1 g/l NH4Cl followed by 4.5 g/l urea in which 5.67 buds per explant were achieved (Table 1).
Table 1
Mean shoot number per explant on medium containing different nitrogen sources.
#Trt
|
Treatment combination
|
NS/ex
|
NB/exp
|
SL(cm)
|
LN
|
RN
|
RL
|
1
|
MS-NH4NO3 + 1.9 g/l KNO3
|
7.33b
|
5.00bc
|
6.23cde
|
4.33a
|
10.33cde
|
10.00b
|
2
|
MS -0.825NH4NO3 + 0.95 g/l KNO3
|
6.33bc
|
4.33cd
|
7.90ab
|
3.67a
|
11.33cd
|
8.96bc
|
3
|
MS -0.825NH4NO3 -0.95 g/l KNO3
|
4.33ed
|
4.00cd
|
4.60fg
|
4.33a
|
7.33fgh
|
8.13cde
|
4
|
MS -0.825 g/l NH4NO3
|
5.00cd
|
5.00bc
|
5.60def
|
4.33a
|
8.33efg
|
7.66de
|
5
|
MS- NH4NO3 + 1 g/l NH4Cl
|
9.33a
|
7.33a
|
8.67a
|
5.00a
|
19.66a
|
12.56a
|
6
|
MS- NH4NO3 + 1.325 g/l NH4Cl
|
2.66ef
|
2.67de
|
3.26gh
|
1.67c
|
6.33ghi
|
7.33ef
|
7
|
MS- NH4NO3 + 1.65 g/l NH4Cl
|
2.00f
|
2.66de
|
2.60h
|
1.66c
|
5.33hi
|
4.73g
|
8
|
MS- NH4NO3 + 2 g/l NH4Cl
|
1.67f
|
3.00d
|
2.06h
|
2.00bc
|
4.00i
|
3.13h
|
9
|
MS -NH4NO3 + 1.5 g/l urea
|
6.67bc
|
6.33ab
|
7.00bcd
|
4.33a
|
10.00cde
|
8.36cde
|
10
|
MS – NH4NO3 + 3 g/l urea
|
7.00b
|
5.67abc
|
7.36abc
|
4.67a
|
11.67c
|
9.23bc
|
11
|
MS – NH4NO3 + 4.5 g/l urea
|
7.00b
|
5.67abc
|
5.67def
|
4.10a
|
14.67b
|
11.73a
|
12
|
MS – NH4NO3 + 6 g/l urea
|
5.00cd
|
4.33cd
|
5.00ef
|
3.5ab
|
9.00def
|
11.70a
|
13
|
MS standard
|
10.00a
|
5.67abc
|
8.56a
|
5.00a
|
16.00b
|
12.86a
|
14
|
MS HF control
|
4.00ed
|
2.67de
|
8.46a
|
4.00a
|
10.00cde
|
6.10fg
|
|
CV (%)
|
19.17
|
22.77
|
14.54
|
24.89
|
14.69
|
10.26
|
Legend
NS/exp-number shoots per explant, NB/explant- number of active buds per explant, SL- shoots length, LN-leaf number per shoot RN- root number per shoot, RL- root length and #Trt-number of treatments.
The result also revealed that there is a significant difference among treatments for the number of active buds which indicated shoot proliferation continues with a similar trend in subsequent sub-culturing as the number of buds is directly proportional to shoot number (Fig. 1). The highest mean shoot number was recorded on medium containing 1 mg/l NH4Cl among the alternatives tasted followed by 3.8 g/l KNO3 and urea at 3 and 4.5 g/l media. On the other hand, a non-significant number of shoots and buds, very short shoots, and roots were recorded on a medium containing a high amount of NH4Cl (> 1 g/l) and urea of more than 4.5 (Table 1). Moreover, it was observed that supplying ammonium chloride with more than 1 g/l and urea above 4.5 g/l resulted in limited root induction and growth accompanied with shoot drying within three weeks of culturing (Fig. 2F and I). This might be due to the toxic effect of high chlorine on plants which prevents the uptake of nutrients by the explants. The number of leaves per shoot is the least affected growth parameter as there is no significant difference among most treatments except for treatments with high NH4Cl and urea. From the result observed three treatments; 1, 5, and 10 (Table 1) at specified levels were observed as potential alternative sources of N2 which can replace NH4NO3 for ginger micro-propagation (Table 1 and Fig. 2).
A study conducted on ginger using double amount (3.8 g/l) of KNO3 in MS media as a sole source of nitrogen indicated that nitrogen in the form of KNO3 significantly improved the proliferation rate of ginger in vitro, in both full and half-strength media. Similar study also reported leaf growth and root formation were also reported better in media devoid of NH4NO3 (Cecilia 2010). Another experiment conducted to substitute NH4NO3 by urea for in vitro regeneration of potato reported that 5 g/l urea resulted in better proliferation and regeneration of potato plantlets which is basically in line with our finding even if the crops are different (Bashara et al. 2021). It is obvious that ammonium nitrate was recently banned in many countries including Ethiopia. Hence tissue culture researchers are globally looking for alternative sources as a replacement specially to compensate ammonium part. Our experiment which included ammonium chloride was the first in its kind to the best of our knowledge which can be adapted to other crops in vitro regeneration protocols.
Root growth and acclimatization
The shoots developed roots spontaneously at the multiplication stage on all medium combinations of salt types and growth regulators. The analysis result showed that there is a highly significant difference among treatments for root number and length. The highest mean number of roots and length were recorded from the medium of KNO3 at 3.8 g/l concentration followed by standard MS medium with 1.65 g/l NH4NO3 and 3 g/l urea (Table 2). Whereas the lowest mean number and length of roots were recorded from the medium in which shoot growth and development were also declined. Media containing 1.325 to 2 g/l of NH4Cl recorded very weak root growth with no significant difference. A similar trend was also observed in root length which decreases when the amount of NH4Cl increases from 1.325 to 2 g/l. The low number and length of roots have affected the growth of shoots in vitro and also affected the acclimatization survival percentage of plants acclimatized from these media. The root and shoot growth inhibition effect observed was directly proportional to the increase in ammonium chloride amount which is probably due to the accumulation of NH+ 4 and chlorine-free ions which could make the media acidic and hence affected nutrient up take and also can act as toxic to plant shoots (Fig. 3b). Study conducted before revealed that the uptake of nitrate ions by plant cells leads to affinity towards an alkaline pH, while NH4 + uptake results in a more rapid shift towards acidity and makes the balance of pH in plants (Hyndman et al. 1982; Edwin et al. 2007). These previous work reports and observations in this experiment tells us the elevated amount of NH4Cl and urea makes the media more acidic which finally limits shoot and root growth for in vitro ginger plantlets production Fig. 3c).
Acclimatization was done after six weeks of culturing directly from the multiplication stage as the plantlets developed roots simultaneously on the multiplication medium. Root development in the multiplication medium of ginger was also reported previously due to enough endogenous auxin hormones to induce roots (Berihu 2018; Genene et al. 2019). Plantlets proliferated together as shoot clumps are detached first after washing out media before acclimatizing. Plants with an average of root number six and above per shoot and medium to long (> 7 cm) root and shoot resulted in 96% survival after 15 days of primary acclimatization (Figs. 3 and 4). Whereas plants with a small shoots, few and short roots failed to achieve enough survival rate after fifteen days of the acclimatization process (Fig. 3b). In this regard plants from a medium with 1.65 and 2 g/l NH4Cl resulted in 15 percent of acclimatization survival due to their weak regeneration. On the other hand, plantlets from 1.325 g NH4Cl and 1.5 g/l urea resulted in 65 and 75% acclimatization survival after 15 days of acclimatization respectively. Plantlets from the remaining 10 media combinations resulted in an average of 94% survival after 15 days. These mediums from which plants are with the highest survival rate were nearly equal with MS standard with and without growth regulators used as standard checks (Fig. 3d). Research reports for the acclimatization survival rate of ginger in vitro regenerated plants are nearly similar to our findings. A study conducted using two cultivars to optimize the protocol for Ethiopian ginger reported an average of 83.5% acclimatization survival (Ayenew et al. 2012). The acclimatization survival rate achieved was also in line with recently published protocol which reported an average of 95% plantlets ex vitro survival for ginger micro-propagation in standard MS media with different growth regulators (Zahid et al. 2021)
Cost benefit analysis
The cost of alternative nitrogen sources has saved costs for in vitro regeneration of ginger with a minimum number of plantlets penalty per culture which can be compensated by purchasing some additional amount of specific alternative salt type. Among the three alternatives tested potassium nitrate followed by ammonium chloride is costly and urea is the cheapest and easily available alternative source of nitrogen (Table 2). Urea at $0.88/kg moment price to use 3 g/l amount in one litter of medium has saved 99.75% from an equal amount of NH4NO3 at a pick price of $392 during experiment time in 2021 which might be special case in Ethiopia due to import restriction. Maximum plantlets penalty (30%) per batch culture has also resulted from a medium of urea at a selected amount of 3 g/l which can be compensated by subsequent sub-culturing as it is easily available at a very low cost. Whereas, a kg of ammonium nitrate rarely obtained by high cost ($392/Kg) has only a 30% plantlets advantage over urea, 26.7% over KNO3, and 6.7% to NH4Cl medium. The second better alternative, NH4Cl from which the highest mean shoot number and better growth and also required in a small amount (1 g/l) resulting in statistically non-significant shoot number as compared to standard MS medium (Table 1).
Table 2
Cost and plantlets comparison of alternative sources.
#Item
|
Alternative salts
|
Unit cost ($/kg)
|
Cost saved (%)
|
Average plants/exp
|
#plants penalty (%/exp)
|
1
|
NH4NO3
|
392
|
0
|
10
|
0
|
2
|
KNO3
|
47
|
88
|
7.33
|
26.7
|
3
|
NH4Cl
|
27.45
|
93
|
9.33
|
6.7
|
4
|
Urea
|
0.88
|
99.75
|
7.00
|
30
|