Anther-mediated haploid induction and genetic stability of Phytophthora-resistant backcross lines of Capsicum annuum L

Abstract

Bell pepper is an important vegetable crop from Solanaceae family, which is cultivated and consumed globally due to its delicious, pleasant and nutrient-rich nature. Most of the commercially cultivated bell pepper cultivars are however, highly susceptible to P. capsici. They are recalcitrance to in vitro regeneration, which make the crop improvement efforts difficult through plant tissue culture-based approaches such as double haploid production. In this study, in vitro callus regeneration protocol was established from anthers of two Phytophthora resistant-backcross lines derived from crossing of two highly susceptible bell pepper cultivars (California Wonder and Solan Bharpur) with a highly resistant chilli landrace, CM334.The cultures were initially established in full strength MS medium, supplemented with different concentration of auxin and cytokinin. Maximum callus induction was achieved in MS medium CI2 supplemented with IAA (2 mg/L) and BAP (0.3 mg/L) (41.59 %) at pH 5.8, without heat-shock and cold stress treatments. The light-green and white compact calli were obtained which proliferated and maintained in the half strength MS medium containing 0.1 mg/L Kinetin (pH 5.8). The presence of Phytophthora resistance loci and successful crossings were confirmed through PCR amplification with the closely linked (RGA-C) and pungency −specific (Pun2) molecular markers, and phenotypic evaluations against P. capsici. The haploid nature and genetic stability were revealed through squash staining with 2 % acetocarmin. This study standardized the conditions appropriate for double haploid culture and regeneration of whole plants to speed up the development of Phytophthora-resistant bell pepper varieties.

1. Introduction

Bell Pepper (Capsicum annuum L. var. grossum Sendt; 2n = 2x = 24).) is a major diploid vegetable crop from Solanaceae family, which is native to tropical and subtropical regions of South-Central America, cultivated in both subtropical and temperate regions and consumed worldwide due to its high nutritional and medicinal properties.1, 2 However, the yield of bell pepper is extensively affected by various biotic stresses especially Phytophthora capsici and P. nicotianae,³ which cause foliar blight, crown or root and fruit rot diseases during rainy season, leading to wilting and subsequently whole plant death.⁴ Majority of the bell pepper cultivars were susceptible to these pathogens, and lack of an efficient highly resistant line possessing a broad spectrum of disease resistance in the germplasm and complexity in their inheritance especially for Phytophthora diseases, has made very difficult to improve the existing cultivars.⁵.

The accession Serrano Criollo Morelo 334 (SCM334) is a Mexican chilli landrace, which exhibits a durable and more effective resistant to multiple pathogens including P. capsici, M. incognita, and certain viruses.⁵ Availability of the whole genome sequence information of ‘CM334′ had facilitated several genetic studies and molecular-marker assisted breeding programs to develop the disease resistant varieties of C. annuum L. including chilli pepper and bell pepper against these pathogens around the world.⁶ It has provided efficient marker systems for concerned trait introgression into elite bell pepper cultivars and also helped to elucidate associated genetic and molecular phenomena.⁷.

However, genetic improvement of bell pepper varieties is mostly restricted to traditional and marker-assisted crop breeding approaches, which are time consuming, labour-intensive with lower success rate as the development of homozygous or pure lines possessing desirable traits takes many years.⁸ On the other hands, in vitro anther or ovule culture are the most common and fastest ways to develop the haploid plant and double-haploid (DH) or truly homozygous lines in several crop species.⁹ It also facilitates the use of genome editing, genetic mapping of several genes or QTLs especially those governing the recessive traits,¹⁰ and selection of the recessive mutant lines,¹¹ novel gametoclonal (androclonal) variants¹² and meiotic recombinants with desirable characteristics such as stress tolerance.13, 14.

Haploid embryos or calli induction is driven by switching of the microspores from gametophytic pathway (pollen development) to sporophytic pathway under in vitro conditions, which often required prolonged heat or cold pretreatment.15, 16 It usually results embryo like structures (embryoids) which subsequently give rise to the whole plants with haploid chromosome number. However, only a few pollen grains in an anther can enter the embryogenic pathway.¹⁷ Most of the C. annuum L. varieties including bell peppers have poor regeneration potentials,¹¹ which limit the applications of various biotechnological interventions. It also hampers the development of doubled-haploid (DH) lines in bell pepper through anthers or pollen culture.18, 19 Therefore, this investigation aimed to establish the in vitro haploid cultures of two backcross lines bearing Phytophthora resistant trait and manipulation of the culture medium to alter the morphogenetic potential of microspores in the cultured anthers and optimized tissue culture conditions necessary to enhance further genetic improvements of such lines.

2. Materials and methods

2.1. Plant materials

The plant materials were grown in the glasshouse conditions, where a chilli landrace, CM334 was crossed as male parent with the bell pepper cultivars, ‘California Wonder’ and ‘Solan Bharpur’ to obtained two types of F₁ hybrid plants. The crossing was performed in the early morning (6.00 am to 8.00 am) followed by begging with butter paper to avoid unnecessary mating. The F₁ plants were further crossed with the recipient or recurrent parents to obtain the two different BC₁F₁ generations (Fig. 1e and 1f). Plant materials were grown under favorable conditions comprising 16 h/8h day/night (34 °C/25 °C).

Fig. 1.

Disease screening of the parents, California Wonder (a) and Solan Bharpur (b) and their backcross line, (CW x CM334) x CW (c) and (SB x CM334) x SB (d), obtained using crossings scheme (e) with have blocky shaped fruits (f).

Screening of Parents and BC₁F₁ generations for disease resistance was performed by using Phytophthora capsici isolate Pc_UHF (GenBank accession no. OR835560), and following the method as described by Kumar et al. (2024b) and recorded the disease severity using a scale as described by Kim et al.²⁰: 0 = no visible symptoms, 1 = small rots on the base of the stem, to up to 30 % of the plant affected, 2 = 31–50 % of the plant affected; 3 = 51––70 % of the plant affected, 4 = 71–90 % of the plant affected, 5 = dead plant, 100 % of the plant affected.

Selected BC₁F₁ plants possessing a high level of Phytophthora blight resistance similar to that of resistant chilli parent, CM334 (Fig. 1c and 1d), were used for molecular evaluation as well as establishment of the haploid cultures. Furthermore, selected BC₁F₁ plants were evaluated for seven morphological characteristics including fruit weight (gm), fruit length (cm), fruit width (cm), fruit shape, pericarp thickness, leaf pubescence and number of lobes per fruit along with parental lines by following DUS guidelines (PPV & FRA 2001).

2.2. Molecular evaluations

Fresh leaf samples were collected from the parents and both the backcross lines at 6 leaf stages and used for genomic DNA isolation using a modified CTAB method.²¹ The DNA samples were used as templates for PCR amplification with pungency-linked Pun2 (5′- ATGGCTTTTGCATTACCATCA-3′/5′-CCTTCACAATTATTCGCCCA-3′)²² and P. capsici resistance linked RGA-C markers (5′-ATGGGAAGCAAGTATTCCAA-3′/ 5′-AGTTTCCACA-GCACATCACC-3′).²³ The PCR amplifications were carried out in an Applied BioSytem PCR thermal cycler using the 15 μl reaction mixtures containing 100 ng template DNA, 10 pmole of each of two primers (forward and reverse), 2.5 mM of dNTPs, 15 mM of MgCl2, 10X PCR buffer and 3 U/μl Taq polymerase (GeNei, Bangalore).The PCR amplification conditions were consisted of initial denaturation at 95 °C for 3 min, followed by 35 cycles each with denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and chain extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR amplicons were subjected into gel electrophoresis using 2 % agarose stained with ethidium bromide (10 mg/mL), and run with 1X TAE buffer at 65-80Volts for 60–90 min. The PCR amplicons resolved on agarose gel were visualized through UV exposure in the SynGene Bio Imaging System.

2.3. Sterilization of plant materials

The closed floral bud from selected backcross plants were selected based on various morphological parameters as described previously.²⁴ In details, flower buds of different size ranges (3 mm-7 mm) 45 days after transplanting, were selected with the help of Vernier caliper. It included flower buds of different stages of the microsporogenesis in anthers ranging from microspore mother cells to uninucleate microspore stages. Seeds from the ‘California Wonder’ (bell pepper parent) were used for comparative study to establish in vitro callus culture and sterilized along with floral buds in the present study.

Successful sterilization is based on the kind of sterilising agent used, its concentration, and the duration of the sterilization treatments in the explants. The plant materials were subjected to sterilisation as it was harvested from the pots and microbially contaminated. An entire tissue cultured flask/jar can become contaminated by even a small amount of fungal or bacterial infection. Young flower buds were washed firstly under running tap water with a pinch of carbendazim and teepol. The explants were then treated with 1 % NaOCl at different time intervals as described in the Table 1, and then rinsed with autoclaved distilled water thrice under aseptic conditions.

Table 1.

Effects of different sterilization treatments on explant survival.

Sr. no.	Carbendazim (%)	Duration (min)	Sodium hypochlorite (%)	Duration (min)	Mean percent uncontaminated explants (%)	Mean percent survival (%)
ST1	Control	−	−	−	0.000 (0.00e)	0.000 (0.000e)
ST2	0.2	5	1.0	1	52.72 (46.56d)	31.550 (34.17d)
ST3	0.2	5	1.0	2	65.82 (54.22c)	49.273 (44.584b)
ST4	0.2	5	1.0	3	79.11 (62.81b)	81.05 (64.210c)
ST5	0.2	5	1.0	4	88.75 (70.53a)	61.38 (51.594a)
ST6	0.2	5	1.0	5	91.47 (73.05a)	41.1 (39.871c)
C.D. (5 %) ± S.E. (mean)					2.661 ± 0.95	1.682 ± 0.70

* Angular transformed value in the parenthesis.

** C.D.-Critical Difference.

***S.E.-Standard Error.

2.4. Media preparation and sterilization

MS media augmented with various concentration of plant growth regulators, sugar as carbon and additional supplements as described in the Table 2, Table 3, Table 4, adjusted at an optimal pH between 5.7 and 5.8 and solidified using agar 7–8 g/litre was prepared. The media was dispensed into the tissue culture vials or tubes with an average volume of 20–25 mL and sterilized by autoclaving at 15 psi and 121 ⁰C for 20 min.

Table 2.

Different Callus Induction (CI) media used for anther culture in this study.

Medium Code	Composition	Reference	Callus Induction (%)	Average number of days for callus induction	Observations
Control	MS medium without growth regulator		0.000 (0.000d)	−	No growth
CI1	MS + BAP (1 mg/L) + GA3 (2 mg/L) + AgNO3 (5 mg/L) + Calcium Pentothenate (2 mg/L) + Vitamin B12 (2 mg/L) + sucrose (3 %) + 0.7 % agar	This study	17.000 (24.350b)	57	Brown and compact callus
CI2	MS + IAA (2 mg/L) + BAP (0.3 mg/L) + sucrose (3 %) + 0.7 % agar	Vijayan et al, (2023)	41.597 (40.162a)	64	Yellowish green and compact callus
CI3	MS + NAA (4 mg/L) + BAP (0.50 mg/L) + AgNO3 (15 mg/L) + Biotin (0.05 mg/L) + sucrose (3 %) + 0.7 % agar	Sahana et al. (2024)	33.000 (35.061a)	49	Yellow and compact callus
CI4	B5 + NAA (4 mg/L) + BAP (1 mg/L) + sucrose (3 %) + AgNO3 (15 mg/L) + 0.7 % agar	Sahana et al. (2024)	0.000 (0.000d)	−	−
CI5	MS + NAA (4 mg/L) + BAP (0.5 mg/L) + AgNO3 (7 mg/L) + Maltose (3 %) + 0.7 % agar	Doganguzel et al. (2021)	8.340 (16.785c)	44	Green and compact callus
CI6	MS + 2,4-D (0.1 mg/L) + Kinetin (0.1 mg/L) + sucrose (3 %) + Biotin (0.1 mg/L) + 0.7 % agar	Grozeva et al. (2021)	0.000 (0.000d)	−	−
CI7	MS + BAP (2 mg/L) + NAA (0.5 mg/L) + sucrose (3 %) + 0.7 % agar		0.001 (0.000d)	−
C.D. ± S.E. (mean)			0.260 ± 0.081

* Angular transformed value in the parenthesis.

** C.D.-Critical Difference.

***S.E.-Standard Error.

Table 3.

Different Callus Induction (CI) media used for diploid culture in this study.

Medium Code	Composition	Reference	Callus Induction (%)	Average number of days for callus induction	Observations
Control	MS medium without growth regulator		0.000 (0.000b)	−	No growth
CI1	MS + BAP (1 mg/L) + GA3 (2 mg/L) + AgNO3 (5 mg/L) + Calcium Pentothenate (2 mg/L) + Vitamin B12 (2 mg/L) + sucrose (3 %) + 0.7 % agar	This study	0.000 (0.000c)	−	−
CI2	MS + IAA (2 mg/L) + BAP (0.3 mg/L) + sucrose (3 %) + 0.7 % agar	Vijayan et al, (2023)	12.400 (10.110a)	63	Light green and compact
CI3	MS + NAA (4 mg/L) + BAP (0.50 mg/L) + AgNO3 (15 mg/L) + Biotin (0.05 mg/L) + sucrose (3 %) + 0.7 % agar	Sahana et al. (2024)	0.000 (0.000c)	−	−
CI4	B5 + NAA (4 mg/L) + BAP (1 mg/L) + sucrose (3 %) + AgNO3 (15 mg/L) + 0.7 % agar	Sahana et al. (2024)	0.000 (0.000c)	−	−
CI5	MS + NAA (4 mg/L) + BAP (0.5 mg/L) + AgNO3 (7 mg/L) + Maltose (3 %) + 0.7 % agar	Doganguzel et al. (2021)	0.000 (0.000c)	−	−
CI6	MS + 2,4-D (0.1 mg/L) + Kinetin (0.1 mg/L) + sucrose (3 %) + Biotin (0.1 mg/L) + 0.7 % agar	Grozeva et al. (2021)	0.000 (0.000c)	−	−
CI7	MS + BAP (2 mg/L) + NAA (0.5 mg/L) + sucrose (3 %) + 0.7 % agar		39.6 (37.983b)	58	Green and compact callus
C.D. ± S.E. (mean)			2.10 ± 0.699

* Angular transformed value in the parenthesis.

** C.D.-Critical Difference.

***S.E.-Standard Error.

Table 4.

Different Callus Proliferation (CP) media for anther culture used in this study.

Code	Medium combination	Reference	Callus proliferation (%)
CP1	Full strength MS + BAP (1 mg/L) + GA3 (2 mg/L) + AgNO3 (5 mg/L) + sucrose (3 %) + 0.8 % agar	This study	21.620 (27.699b)
CP2	Full strength MS + BAP (1 mg/L) + sucrose (3 %) + 0.8 % agar	This study	0.000 (0.000c)
CP3	Half Strength MS + kinetin (0.1 mg/L) + AgNO3 (7 mg/L) + sucrose (2 %) + 0.8 % agar	This study	52.130 (46.221a)
CP4	Half strength MS + BAP (0.1 mg/L) + sucrose (3 %) + agar (0.7 %)	Sahana et al. (2024)	0.000 (0.000c)
C.D. (5 %). ± S.E. (mean)			15.11 ± 0.59

* Angular transformed value in the parenthesis.

** C.D.-Critical Difference.

***S.E.-Standard Error.

2.5. Establishment of anther cultures

Floral buds from BC1F1 lines were excised to isolate the anthers (4–4.5 mm) using sterile scalpel blade, and separated the anthers from filaments under aseptic conditions. They were inoculated on seven different callus induction (CI) medium derivatives of standard MS along with California Wonder seeds as described supplemented with various concentrations of plant growth regulators (Table 2, Table 3). Inoculated cultures were incubated in the dark for one to two weeks and then transferred to 12 h/8h day/night photoperiod (25–––30 μmol m⁻² s⁻¹ irradiance), at 25 °C and 60–70 % relative humidity. Development of the callus first week onward from inoculation was analysed up to eight weeks, and callus induction (CI) frequency was calculated using the following formula²⁵:

$$\mathit{Callusinductionfrequency}=numberofanthersorseedswithcallus/totalnumberofanthersorseedsinoculated\times 100$$

Callusogenic anthers were sub-cultured after 45–70 days interval for proliferation in the different callus proliferation (CP) media as described in the Table 4, and callus proliferation frequency was calculated. The observed data were further analyzed in a completely randomized design (CRD) through one-way analysis of variance (ANOVA) and Duncan’s multiple range test using the OPSTAT software to test the significance of mean differences (P < 0.05).26, 27

2.6. Chromosome estimation and meiotic analysis

Cytological studies was performed on floral buds of different sizes to deduce the genetic stability of a plant.²⁸ Young flower buds of different developmental stages were collected in the early morning (6.00–9.00 am) from BC₁F₁ plants cultivated in the glass house. They were fixed in the Carnoyʼs fixative solution comprising ethanol: chloroform: acetic acid (v/v/v) (6:3:1) for 24 h. The fixed flower buds were then preserved in the absolute ethanol and stored at 4 °C until used. Fixed flower bud of different sizes were smeared using standard acetocarmine staining technique.29, 30 Approximately 15–25 freshly prepared slides from each of the two backcross lines were examined under the compound microscope at 100X (Nikon E-200, Japan).

The > 50 pollen mother cells (PMCs) were examined to ascertain the chromosome number at different meiotic stages including anaphase I and II, metaphase I and Telophase 1. The normal tetrads had 4 equal-sized cells, and those deviated had meiotic abnormalities. Meiotic index (MI)-based on meiosis-II, was estimated by following the formula as described by Love³¹:

$$MI\left(\%\right)=\left[\mathit{numberofnormaltetrads}/totalnumberanalyzedtetrads\right]x100$$

Pollen fertility or viability was determined by examining approximately 500 mature pollen grains mounted in the glycerol–acetocarmine (1:1) mixture. The pollen grains with stained and properly filled nuclei were treated as fertile, while those having shrivelled, empty and unstained pollen nuclei recorded as sterile or non-viable.³²

3. Results

3.1. Development of backcross lines and Phytophthora disease screening

Parent lines (CW, SB and CM334) and backcross lines were screened with the P. capsici isolate Pc_UH for disease resistance. Only ‘CM334′ and both the backcross lines had exhibited high levels of resistance to P. capsici with the disease severity score ranged from 0 to 1,³³ while the CW and SB had shown susceptibility with the disease severity more than 2 in all the tested plants.

3.2. Morphological and molecular evaluations

PCR amplifications with the molecular markers linked to pungency (Pun2) and Phytophthora resistance (RGA-C) had given amplifications only in the resistant parent, CM334, and both the backcross lines (Fig. 2). It has confirmed the successful crossing between parents as well as in between F₁ plants and recurrent parents. These findings were further supported by morphological data of selected BC₁F₁ plants, which were ranged in the bell pepper parents (CW and SB). BC₁F₁ plants from the cross CW x CM334 had 3–4 lobes per fruit with the average weight of 66.94 ± 2.01 g, lack of leaf pubescence and rectangular fruit shape similar to the CW (73.34 ± 0.46 gm). Similarly, BC₁F₁ plants from the cross SB x CM334 had 2–3 lobes per fruit with the average fruit weight of 64.95 ± 5.76gm, lack of leaf pubescence, and trapezoidal fruit shape similar to the SB (67.77 ± 0.67 gm). The average length and width of the BC₁F₁ fruits from the cross CW x CM334 were 5.23 ± 2.65 mm and 4.24 ± 4.76 mm, which were 5.80 ± 0.38 mm and 4.86 ± 0.18 mm in the CW. In the case of BC₁F₁ fruits from the cross SB x CM334, it was 5.12 ± 1.22 mm and 2.91 ± 0.31 deviated from SB having 4.90 ± 0.16 mm long and 3.89 ± 0.15 mm wider fruits. The pericarp thickness of BC₁F₁ fruits from the cross CW x CM334 and SB x CM334 were 4.04 ± 1.94 mm and 3.37 ± 0.40 mm deviated from the CW (4.41 ± 0.16 mm) and SB (4.19 ± 0.17 mm).

Fig. 2.

Banding patterns of pungency marker (Pun2) and a resistant gene analog (RGA-C) in the parents and BC₁F₁ lines on 2% agarose gel.

3.3. Effect of sterilization treatments

Explants were surface sterilized with 0.2 % carbendazim and various concentrations of sodium hypochlorite (NaOCl; 1 %) for different time intervals (Table1). Bacterial contamination was observed initially after three to four days however with increase in sterilization treatments, the level of contamination rate was decreased dramatically. Best results were obtained upon NaOCl treatment for 3 min, which has given 81.05 % survival rate and 79.11 % uncontaminated explants. Maximum number of uncontaminated explants was observed when treated with sodium hypochlorite for 5 min however the survival rate of the explants was reduced proportionally (Table 1, Table 5). Therefore, the best combination was 0.2 % carbendazim treatment for 5 min and NaOCl treatment for 3 min in the ST4. Significant differences were found among the sterilization treatments (except between ST5 and ST6) (C.D. = 2.661 ± 0.95; P-value ≤ 0.05), and also in the mean percent survival (C.D. = 1.682 ± 0.70, P-value ≤ 0.05).

Table 5.

Analysis of Variance (ANOVA) of different parameters evaluated in this study.

Source of Variation	df	Mean sum of Square		df	Mean sum of Square	df	Mean sum of Square
		Mean percent uncontaminated explants	Mean percent survival		Callus Induction		Callus proliferation
Treatment	5	2,182.82**	1,422.21**	6	891.384**	3	1,431.30**
Error	12	2.24	0.894	14	0.019	8	64.429
C.D. (1 %)		3.73	2.34		0.332		21.99
S. E.		0.95	0.70		0.085		0.590

*df: degree of freedom, C.D.-Critical Difference, S.E.-Standard Error.

** P-value < 0.01 significance level.

3.4. Callus induction

Floral buds with average length of 3–5 mm were found more responsive towards callus induction with the highest proportion was recorded in the 4–4.4 mm bud size (25 %), while those with ≥ 6 mm in size, had respond least for the same. Callus is an unorganized mass of cells which was initiated only after 50––60 days of establishment in the present study, and generally dependent on the auxin and cytokinin amalgamation as exogenously supplied growth regulators increase the endogenous levels of cytokinin and auxin, which directly affect callus induction and growth of callus mass. In the case of anther cultures, auxin has high influence on the callus initiation from the anthers, and therefore is mostly used in the callus induction medium. High auxin with low concentration of cytokinin initiated callus growth. Two different auxins (IAA and NAA) were used in different combinations with other growth regulators in which the best result was obtained in medium containing IAA (2 mg/l) with low concentration of BAP (0.3 mg/l). Maximum callus initiation was observed in medium CI2 (41.59 %) followed by CI3 (33 %), CI1 (17 %) and CI5 (Table 2). No sign of callus growth was observed in the callus induction media CI4 and CI6. The rate of callus induction was highest in the CI5 (44 days) and CI3 media (49 days), which took 57 days and 64 days in the CI1 and CI2.

Whitish and Brown callus with compact texture were observed in CI3 medium fortified with NAA (4 mg L^-1and BAP (0.5mgL^-1and CI2 medium containing BAP (1mgL^-1and GA3 (2mgL^-.¹ On the other hand, yellow green and greenish calli with compact texture were obtained for CI2 medium containing IAA (2mgL^-1and BAP (0.3mgL^-1and CI5 medium augmented with NAA (4 mg L-1), BAP (0.5 mg L-1) and maltose (3 %) (Table 2 and Fig. 3a-d). Significant mean differences were observed in the callus induction rate in the all the media composition except between the CI1 and CI6 from the anthers of two different BC₁F₁ lines (C.D. = 0.234 ± 0.085, P-value ≤ 0.05) (Table 2, Table 3, Table 4, Table 5).

Fig. 3.

Establishment of haploid callus culture from the anthers (a-e) and diploid culture from the seeds (f-g).

The increased in the NAA concentration had not promoting effect on callus induction rate from anthers, while too high NAA turned calli into black colour. The IAA (2 mg/L) has more callus induction effect over NAA (4 mg/L), while the use of 2, 4-D was not found beneficial for callus induction from anthers. On the other hand, the use of lower concentration of BAP (0.1–0.5 mg/L) has promoting effect on callus induction over its higher concentration (1 mg/L). BAP was found beneficial and more effective for callus induction over kinetin as no callus formation was observed in the kinetin containing media (CI6).

The days to callus induction indicated a significant effect of maltose on callus induction over sucrose as the earliest callus induction (recorded in the medium CI5 augmented with NAA (4 mg/L), BAP (0.5 mg/L), AgNO₃ (7 mg/L) and 3 % maltose (44 days) followed by CI3 containing NAA (4 mg/L), BAP (0.50 mg/L), AgNO₃ (15 mg/L), Biotin (0.05 mg/L) and sucrose (3 %) (49 days). While the media fortified with IAA (2 mg/L), BAP (0.3 mg/L), and sucrose (3 %) has taken the longest time (64 days) for callus induction. Two alternate concentrations of AgNO₃ (5 and 15 mg/l) were used in the present study and both had induced callus growth and shortened the days required for callus induction as compared to the media without AgNO₃.

In the case of California Wonder seeds, same compositions of growth regulators were used, however the results varied from those observed in the anther cultures. Callus induction from seeds was observed largely in the CI7 medium (BAP (2 mg/L) and NAA (0.5 mg/L)) (39.6 %), which was highest among all the media compositions followed by CI2 (12.4 %) (Table 3). Thus, a considerable variability was recorded in the calli induction rate from the anthers (n-=12) and diploid seeds (2n = 24).

3.5. Callus proliferation

Anther-derived calli were transferred onto callus proliferation media (Table 4) supplemented with low auxin to high cytokinin concentrations to promote rapid cell division and expansion of the callus mass for both haploid and diploid cultures. Both half and full strength MS medium were used with different growth regulators to enhance the proliferation rate. Though anther-derived callus proliferation was observed in both kind of media, proliferation rate of anther-derived calli was higher in the half strength MS medium CP3 (52.13 %) containing kinetin (0.1 mg/L) followed by full strength CP1 medium containing BAP (21.62 %) (Fig. 3e; Table 3). No growth was observed in the CP2 and CP4 media, which indicated the significant role of AgNO3 in the callus proliferation.

Though the proliferation rate of anther-derived calli was increased by both kinetin and BAP in combination with the AgNO3, no shoot regeneration was observed. Calli derived from the ‘California Wonder’ seeds showed highest proliferation in the same culture media (CI7), which was earlier used for callus establishment. No proliferation of the seed-derived calli was observed among the callus proliferation media utilized in the present study. Significant mean differences were observed in the callus proliferation rate among the media combination (C.D. = 15.11 ± 0.59; P-value ≤ 0.05) (Table 4). Moreover, calli derived from the anthers (haploid) were slightly green and compact (Fig. 3d), whereas those derived from diploid seeds were highly greenish in colour and compact (Fig. 3e).

3.6. Chromosome estimation and meiotic analysis

Two BC₁F₁ (backcross) lines derived from the cross California Wonder (female) X CM334 and Solan Bharpur (female) X CM334, showed haploid chromosomal count of n = 12 (2n = 24) as 12 bivalents counted at metaphase 1 stage of meiosis in the PMCs (pollen mother cells) (Fig. 4a-c). Presence of the triads indicated formation of both sterile and fertile pollen formation (Fig. 4d and 4e). Fifty tetrads were examined in this study of which the majority has 4 equal-sized cells (normal), with more than 90 % meiotic index (95 % and 91 % for BC₁F₁ from the cross CW x CM334 and SB x CM334). It indicated that the given backcross lines were mostly meiotically and genetically stable. Few individuals from one of the backcross line (SB x CM334) exhibited variation in tier meiotic indexes at metaphase I, which may be possibly due to the chromosome stickiness, affecting the pollen fertility. The pollen fertility in the both BC₁F₁ lines was recorded high, which ranged from 94.89 % to 96.51 %, with a fewer number of sterile pollens. Mostly, all the pollen grains examined in this study were normal and equal in the shape and size (Fig. 4f).

Fig. 4.

Meiotic chromosomal behavior at different stages of microsporogenesis (a-e), and estimation of pollen viability (f).

4. Discussion

Peppers from the C. annuum L. are considered recalcitrant to in vitro androgenesis associated with reduction in the embryo induction frequency from anthers and low regeneration rate from the embryogenic calli that suggested being due to the high ethylene activity during callus induction phase.11, 34 AgNO3 is the most used ethylene inhibitors in the anther culture.³⁵ Haploidy induction in bell peppers was firstly employed though anther culture in 1973,³⁶ and reproducible anther culture was obtained in 1981 by Dumas de Vaulx et al..³⁷ Subsequently, numerous attempts were made to determine the various factors including tissue culture media, concentration and types of plant growth factors,³⁸ developmental stage of microspores, etc., significantly affecting haploid induction potential of the anthers. Kim and Lim³⁹ optimized the conditions for the formation of leaf segment-derived calli from the genome-sequenced hot pepper (‘CM334′) and bell pepper (‘Dempsey’).

Notably, Mangal and Srivasatava²⁴ had described various morphological characteristics such as shape, size and colour of flower bud correlated with the stage of microspore development inside the anthers. The 6–7 mm (mm) long flower bud with nearly 450um green anthers, purple pigmentation at the apical end, and slight variation in the length of calyx and corolla were indicated late uninucleate to early binucleate microspore stage which is the most conducive for successful androgenesis and sensitive to external stimuli.⁴⁰ Sahana et al.² observed average flower buds size and anther length of 5.39 mm and 2.75 mm in Orobelle, 4.8 mm and 2.71 mm in Bomby, which had uninucleate pollen grains.

Auxin is a dynamic phytohormone crucial for the growth and development of callus, whereas the alteration of endogenous cytokinin plays a key role in cell aggregation, resulting in a more compact callus structure, as well as promoting cell division that leads to the growth of shoots and stems. Typically, elevated levels of auxin coupled with reduced concentrations of cytokinin in the culture medium enhance cell proliferation and callus formation.⁴¹ The interplay between auxins and cytokinins regulates cell division, which in turn boosts the activity of cdc2/cdk2-like kinase, leading to increased callus production.⁴² The application of external plant growth regulators (PGRs) also influences the internal levels of enzymes and plant hormones, subsequently impacting the color, texture, and characteristics of the callus.⁴³.

In the present study, introgression of the P. capsici resistance linked QTLs was achieved through marker-assisted backcross breeding from ‘CM334′ into two elite bell pepper cultivars, ‘California Wonder’ and ‘Solan Bharpur’. Selected backcross lines with a high degree of resistance to Phytophthora blight, lack of pungency as confirmed by linked molecular markers and blocky fruit shape were used to establish the anther culture for further genetic improvement through biotechnological interventions such as double haploids, and also examined for genetic stability. Various types of growth regulators with different concentrations and combinations were used for callus initiation from anthers and seeds.

The optimal conditions used to obtain the anther-derived calli were determined such as the sterilization treatment (0.2 % carbendazim for 5 min and NaOCl treatment for 3 min), callus initiation medium (MS + IAA (2 mg/L) + BAP (0.3 mg/L) + sucrose (3 %) + 0.7 % agar), and callus proliferation medium (Half Strength MS + kinetin (0.1 mg/L) + AgNO3 (7 mg/L) + sucrose (2 %) + 0.8 % agar), which gave optimal results. Flower buds of an average length 3–5 mm and calyx covering approximately 70–80 % of total bud length, had resulted highest callus induction, which were mostly at late-uninucleate² or early binucleate stage, determined as the most conducive stage in bell pepper.⁴⁴ The composition of the culture medium was found highly dependent on the genotype of the donor plant.⁴⁵.

Callus initiation observed after 50––60 days of establishment without subjected the flower buds to cold pretreatment⁴⁶ or heat shock treatments to fresh anther cultures as previously suggested.47, 48 It opposed the previous reports indicating androgenesis promoting effects of cold pretreatment and heat shock treatments.49, 50, 51 The callus initiation rate was extensively promoted by incubating the fresh anther cultures under dark conditions for first 14 days, followed by incubating under normal day/light (16 h/8h) conditions at 24 °C and 55–60 % relative humidity. Low concentration of NAA over IAA in combination with BAP over Kinetin, with or without silver nitrate has significant implications on successful callus induction.⁵² An excellent callus induction was observed at 2 mg/L of IAA and 4 mg/L of NAA after a culture period of 7–9 weeks, which was in close conformity with the Manisha et al.⁵⁰ findings. Overall, IAA showed higher callus induction than NAA and 2, 4-D (Table 2). The use of maltose over sucrose found as more conducive carbon source, which shortened the time required for callus induction, while no major effects found associated with the use of biotin in the callus induction and proliferation media.⁵².

Grozeva and Nankar⁴⁵ assessed in vitro androgenic response of two prominent Bulgarian pepper varieties in five different incubation period and regeneration mediums with varying concentration of kinetin (0.1, 0.2 and 0.3 mg L-1) and zeatin (0.25 mg L-1 0.5 mg L-1) in combination with the indole acetic acid (IAA) (0.1 mg L1). Prolonged anthers incubation on induction medium had a positive effect on embryo formation and plantlet regeneration as the maximum embryo formation and plant-regenerants was recorded in the variety Stryama after 18 days with medium supplemented with 0.1 mg L-1 kinetin. Embryo induction and plantlet regeneration in the variety Zlaten Medal 7 were highest after 16 days and 20 days in the control media without any growth regulators.

Furthermore, a great variation was observed in the callusogenic potentials of anthers derived from two backcross lines as the BC₁F₁ line from the cross CW x CM334 had more callusogenic anthers (22 %) than the other BC₁F₁ line from the cross, SB x CM334 (13.3 %). It supported the previous reports of significance of the genotype on successful anther cultures of recalcitrant peppers.¹¹ Ercan et al.⁵³ revealed distinct embryogenic responses by two different pepper genotypes in response to seasonal effects on anthers such as the highest embryogenesis was recorded in the summer season for Kekova cultivar and in winter for Sera Demre 8 cultivar, especially when anthers from 4-month-old plants. It indicated that the high embryogenic potential in the anthers was obtained from the older plants.

The in vitro response of majority of the callusogenic anthers was non-embryogenic callus with the exception of embryogenic callus formation without shooting and plantlets formation such as 20 % and 12 % of embryogenic anthers of selected BC₁F₁ progenies from the cross CW x CM334 and SB x CM334. It was possibly due to both the genetic and environmental factors. Higher callusogenic potential does not mean better morphogenesis ability because of lack of direct embryogenesis and regeneration from anther cultures of majority of the pepper genotypes.54, 55 A large proportion of anthers from both the backcross lines were identified non-responsive.³⁷ No successful plant regeneration was accomplished from anther derived microspore calli in this study despite using the various media compositions. However, the presence of green calli in some of the anther cultures indicated their embryogenic growth, which indicated a reasonable possibility of getting complete plantlet regeneration from such calli.

The ploidy level of the microspore was determined using the standard squash technique, which confirmed high genetic stability of Phytophthora resistant backcross lines. Both triads and tetrads were observed in the cytological examinations, which may affect the pollen fertility. Two BC₁F₁ (backcross) lines derived from the cross CW x CM334 and SB x CM334, showed haploid chromosomal count of n = 12 (2n = 24) as 12 bivalents counted at metaphase 1 stage of meiosis in the PMCs (pollen mother cells) (Fig. 4a-c), which is in conformity with the previous reports.56, 57 No major chromosomal aberrations were recorded during meiosis, though formation of triads was observed along with regular tetrad formation, normal microsporogenesis and PMCs with equal chromosome numbers in both BC₁F₁ lines, reinforcing their genetically stability.

Presence of triads indicated the formation of both sterile and fertile pollen formation (Fig. 4d and e). Previous studies also showed the presence of certain chromatin stickiness from early prophase I to telophase II (T-II), associated with partial or often complete clumping of bivalents/chromosomes, and chromosome elimination during metaphase stage of meiosis in C. annuum L., C. frutescens L. and C. pubescens.⁵⁸ Certain abiotic stresses such as chilling, and pathogens including viruses may alter the cytological processes in plants including meiosis, and also results somatic polyploidy as suggested in the previous reports, which supported the presence of meiotic irregularities (such as univalents, laggards, bridges, micronuclei, etc.) in the virus infected C. annuum and C. pendulum plants.57, 59.

Various heritable factors with quantitative inheritance determine the successful induction of haploids from anthers such as androgenic potential of F₁ progenies is dependent on their parent.⁶⁰ Backcross progenies had relatively higher androgenesis than F₁, which indicated genetic background had large effect on angrogensis in the BC₁F₁ progenies.⁶¹ Furthermore, regeneration potential of the bell pepper varieties is relatively very low such as 5–15 % for ‘California Wonder which restrict the application of haploids and double haploids.³⁶ Genetic variability between genotypes and anther developmental stages had a considerable influence on the callus induction and proliferation rates of their progenies, which possibly affect the pepper regeneration rates and associated morphologic responses.

5. Conclusion

This study highlights the potential of anther culture and microspore embryogenesis as an advanced breeding method for rapid production of superior homozygous lines, unique genetic recombinants, improving selection efficiency especially for recessive mutations and facilitates the use of plant genome engineering approaches for genetic improvements of recalcitrant Capsicum annuum cultivars. The application of high auxin (IAA and NAA) and low cytokinin (BAP) had significantly increased the callus induction and growth in the haploid culture, whereas the callus induction media supplemented with high BAP (2 mg/L) and low NAA (0.5 mg/L) had significantly increased the callus induction rate in the diploid culture. Two improved anther culture media (CI2 and CI3) were established based on their ability to enhance the callus formation, embryogenesis and call mass proliferation in haploid culture and one media (CI7) for diploid callus culture. This study indicated that culture media and genotype of the donor plants had significantly effect on callus formation and proliferation efficiency. Two BC1F1 lines exhibiting resistance to P. capsici were found genetically stable with high pollen viability.

Funding statement

No external funding was received for this work.

CRediT authorship contribution statement

Surender Kumar: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Aayushee Thakur: Writing – review & editing, Validation, Data curation. Munish Sharma: Writing – review & editing, Visualization, Data curation. Neha Thakur: Visualization, Resources, Investigation. Sanjeev Kumar: Writing – review & editing, Validation, Investigation, Conceptualization. Anupama Singh: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.