Anther Culture- A total of 13538 anthers form ten genotypes cultured on six different media combinations/protocols. Approximately 200 to 250 anthers were cultured per genotype- per media combination/protocol (Table 1). Out of six, only four media combinations responded for one step of direct embryogenesis including shed-microspore clture protocol and two-step anther culture protocol. CAEI-1 media showed the highest embryo formation frequency (40.79%) as well as the highest plant regeneration frequency (20.62%), while CAEI-6 media showed the lowest embryo formation (0.81%) and plant regeneration frequency (0.41%) (Figure-3). Media codes CAEI-2 & CAEI-3 did not respond to one-step direct embryogenesis. However, shed-microspore culture protocol (media code -CAEI-5) also responded for microspore embryogenesis with 2.63% embryo formation frequency and plant regeneration frequency of 1.4%. The two-step anther culture protocol responded with 1.39% embryo formation frequency and plant regeneration frequency of 0.65%. Compared to these shed-microspore and two-step anther culture protocol, our media combination resulted with very high frequency of embryogenesis as well as plant regeneration frequency.
Embryo harvesting and Plant development -We were very keen at the time of embryo harvesting, and confirmed the androgenic origin of all harvested embryos; there is no involvement of somatic cells in embryo development. When embryo was observed under microscope at 4X magnification at that time the anther wall was fully opened and a microspore developed in to an embryo and few microspores besides the developed embryo also present in that anther and those all are in process to develop as an embryo. This microscopic observation confirms that embryos were developed from microspores and not from somatic cells. We gently detached embryo from anther and inoculated on plant conversion media. Detaching embryo from anther needs very skillful hand, because, if the embryo got broken during harvesting process, then chances of broken embryo to convert in to normal plant are very less. More care was taken while harvesting embryos from anther culture protocols, whereas in case of shed-microspore culture protocol microspores bursted in liquid layer and converted in to embryos. We have harvested a total of 1020 embryos derived from all genotypes and developed on four different media combinations/protocols (CAEI-1, CAEI-4, CAEI-5, CAEI-6). Out of 1020 only 516 embryos developed in to normal plants with an average frequency of 50.85%, remaining embryos were abnormal, hence discarded.
Table 1
Frequencies obtained during media optimization for one-step direct embryogenesis in Capsicum annuum
|
Genotype Numbers
|
|
Media Codes
|
IC-1120
|
IC-1121
|
IC-1122
|
IC-1123
|
IC-1124
|
EC-2212
|
EC-2213
|
EC-2214
|
EC-2215
|
EC-2216
|
No of anthers cultured
|
No of Embryos germinated
|
No of plants developed
|
No of DH plants
|
Embryo germination frequency
|
Plant regeneration frequency
|
Anther to DH frequency
|
CAEI-1
|
210
|
212
|
220
|
225
|
225
|
252
|
232
|
210
|
205
|
240
|
2231
|
910
|
460
|
186
|
40.79%
|
20.62%
|
8.34%
|
CAEI-2
|
220
|
232
|
210
|
210
|
265
|
250
|
205
|
220
|
240
|
210
|
2262
|
0
|
0
|
0
|
0.00
|
0.00
|
0.00
|
CAEI-3
|
242
|
240
|
210
|
215
|
240
|
210
|
210
|
245
|
215
|
214
|
2241
|
0
|
0
|
0
|
0.00
|
0.00
|
0.00
|
CAEI-4
|
252
|
245
|
225
|
212
|
245
|
232
|
225
|
220
|
210
|
240
|
2306
|
32
|
15
|
6
|
1.39%
|
0.65%
|
0.26%
|
CAEI-5
|
205
|
265
|
205
|
252
|
255
|
230
|
225
|
210
|
200
|
232
|
2279
|
60
|
32
|
13
|
2.63%
|
1.4%
|
0.57%
|
CAEI-6
|
212
|
205
|
265
|
242
|
220
|
220
|
210
|
225
|
205
|
215
|
2219
|
18
|
9
|
5
|
0.81%
|
0.41%
|
0.23%
|
Total
|
1349
|
1399
|
1335
|
1356
|
1450
|
1394
|
1307
|
1330
|
1275
|
1351
|
13538
|
1020
|
516
|
210
|
7.53%
|
3.81%
|
1.55%
|
Media combinations / protocols-
- CAEI-1 – MS medium with vitamins + 0.1mg/lit TDZ + 0.5mg/lit IAA + 30gm/lit Sucrose + 3gm/lit Gelrite®, pH-5.8
- CAEI-2 – MS medium with vitamins + 0.5mg/lit BAP + 1mg/lit NAA + 100gm/lit Sucrose + 3gm/lit Gelrite®, pH-5.8
- CAEI-3 – MS medium with vitamins + 1mg/lit 2,4-D + 0.1mg/lit NAA + 100gm/lit Sucrose + 3gm/lit Gelrite®, pH-5.8
- CAEI-4 – Induction medium – C medium supplemented with 0.01 mg/lit kinetin + 0.01 mg/lit 2,4, D +30gm/lit Sucrose + 8gm/lit Agar, pH-5.9
Regeneration medium – R medium supplemented with 0.1 mg/lit kinetin 30gm/lit Sucrose + 8gm/lit Agar, pH-5.9 (Dumas de Vaulx et.al 1981) and (Para-Vega et.al 2016)
- CAEI-5 – Upper layer liquid medium (Nitsch medium with vitamins + 2.5 muM Zeatin + 5 muM IAA + 20gm/lit Maltose)
Under layer solid medium (Nitsch medium with vitamins +20gm/lit Maltose + 10gm/lit activated charcoal+ 8gm/lit Micro Agar®, pH-5.8 (Supena et.al.2006)
- CAEI-6 – MS medium with vitamins + 1mg/lit BAP + 1mg/lit Kinetin + 0.5mg/lit IAA + 30gm/lit Sucrose + 8gm/lit Agar, pH-5.8
Table 2
Genotype wise plant regeneration frequency obtained on CAEI-1 media combination
Genotype Numbers
|
No. of Anthers Cultured
|
No. of Plants Regenerated
|
Plant Regeneration Frequency
|
IC-1120
|
210
|
80
|
38.10%
|
IC-1121
|
212
|
28
|
13.21%
|
IC-1122
|
220
|
35
|
15.91%
|
IC-1123
|
225
|
45
|
20%
|
IC-1124
|
225
|
42
|
18.67%
|
EC-2212
|
252
|
18
|
7.14%
|
EC-2213
|
232
|
60
|
25.86%
|
EC-2214
|
210
|
45
|
21.43
|
EC-2215
|
205
|
44
|
21.46
|
EC-2216
|
240
|
63
|
26.25
|
Total
|
2231
|
460
|
20.62
|
Genotypic Variation in Androgenesis- We also studied genotype-wise frequencies and found that genotype number IC-1120 showed the highest plant regeneration frequency (6.41%), and genotype number EC-2214 showed the lowest plant regeneration frequency (2.41%). When we observed data of anthers cultured on the highest responding media, i.e., CAEI-1 (Table-2), the highest plant regeneration frequency is observed in genotype number IC-1120 (38.1%), which is relatively higher compared to reported frequencies. The lowest plant regeneration frequency is observed in genotype number EC-2212 (7.14%), which is also suitable for commercial DH production. This data reveals that there is variation in plant regeneration frequencies of different genotypes; this is predominantly regulated by the expression of transcriptional factors, namely BABY BOOM (Boutilier et al., 2002), LEAFY COTYLEDON1 (LEC1; Lotan et al., 1998), LEC2 (Stone et al., 2001), and FUSCA3 (To et al., 2006), present in the genome of plants.
Table 3
Genotype wise plant regeneration frequency obtained on CAEI-6 media combination
Genotype Numbers
|
No. of Anthers Cultured
|
No. of Plants Regenerated
|
Plant Regeneration Frequency
|
IC-1120
|
212
|
4
|
1.9%
|
IC-1121
|
205
|
0
|
0
|
IC-1122
|
265
|
0
|
0
|
IC-1123
|
242
|
1
|
0.4%
|
IC-1124
|
220
|
0
|
0
|
EC-2212
|
220
|
0
|
0
|
EC-2213
|
210
|
2
|
1%
|
EC-2214
|
225
|
0
|
0
|
EC-2215
|
205
|
1
|
0.5%
|
EC-2216
|
215
|
1
|
0.5%
|
Total
|
2219
|
9
|
0.4%
|
On the other hand, when we observed the data of the lowest responding media, i.e., CAEI-6 (Table-3), and found that the highest plant regeneration frequency is observed in genotype number IC-1120 (1.9%). The lowest plant regeneration frequency is observed in genotype number IC-1123 (0.4%), and five genotypes (IC-1121, IC-1122, IC-1124, EC-2212, and EC-2214) did not respond for embryogenesis on this media combination. These results suggest that media code CAEI-6 is low-responding and highly genotype-dependent.
Ploidy Analysis- We have analyzed the ploidy level of 516 regenerated plants and found that histograms of 210 plant samples resemble histograms of standard diploid samples and are considered DHs. As all plants were regenerated from microspores, we observed spontaneous chromosome doubling in 40.70% of the total analyzed plants (Figure-6 & Table-4). Spontaneous chromosome doubling frequencies among all ten genotypes ranges between 23.26–53.42%, highest rate of spontaneous chromosome doubling observed in genotype number-EC 2213 and genotype number-IC 1124 showed lowest rate of spontaneous chromosome doubling. Samples from 300 plants showed histograms on the half scale of standard diploid considered as haploid as it contains only half nucleic acid content (one set of chromosomes) compared to standard diploid. Six plants considered as mixoploids as histograms of those samples showed two peaks, one resembles to standard scale as well as on half scale of standard diploid sample. As per analysis of histogram of mixoploid samples, we concluded that it contains diploid/doubled haploid as well as haploid cells. Besides artificial chromosome doubling using anti-mitotic agents, the spontaneous doubling of chromosomes in plant cells takes place due to three mechanisms- Nuclear fusion, Endoreduplication, and Endomitosis (Segui-Simarro & Nuez, 2008). Out of these three mechanisms of spontaneous chromosome doubling, 90% times endoreduplication will takes place. We have not done any artificial chromosome doubling using any anti-mitotic agent such as colchicine, oryzaline, triflurine etc.
Table 4
– Data of ploidy analysis with Genotype wise spontaneous chromosome doubling frequency
Genotype Numbers
|
No. of Plants Analyzed
|
No. of DH Plants
|
No. of Haploid Plants
|
No. of Mixoploid Plants
|
Spontaneous chromosome doubling Frequency
|
IC-1120
|
89
|
38
|
50
|
1
|
42.70
|
IC-1121
|
31
|
13
|
17
|
1
|
41.94
|
IC-1122
|
39
|
15
|
24
|
0
|
38.46
|
IC-1123
|
47
|
15
|
31
|
1
|
31.91
|
IC-1124
|
43
|
10
|
33
|
0
|
23.26
|
EC-2212
|
19
|
5
|
14
|
0
|
26.32
|
EC-2213
|
73
|
39
|
32
|
2
|
53.42
|
EC-2214
|
56
|
20
|
36
|
0
|
35.71
|
EC-2215
|
48
|
23
|
25
|
0
|
47.92
|
EC-2216
|
71
|
32
|
38
|
1
|
45.07
|
Total
|
516
|
210
|
300
|
6
|
40.70
|
Plant acclimatization- We planted all 210 confirmed DH plants in sterile cocopeat and 205 plants were survived after acclimatization process with survival index of 97.61%. During planting process, only well rooted and well grown plants were selected. All process of plant acclimatization was strictly followed to achieve maximum survival rate.