Efficient microspore embryogenesis in cauliflower (Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in breeding programme

Scientia Horticulturae 216 (2017) 83–92

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Efficient microspore embryogenesis in cauliflower (Brassica oleracea var. botrytis L.) for development of plants with different ploidy level and their use in breeding programme R. Bhatia ∗ , S.S. Dey, Shritika Sood, Kanika Sharma, Chander Parkash, Raj Kumar ICAR-Indian Agricultural Research Institute, Regional Station, Katrain, Kullu, Himachal Pradesh, 175129, India

a r t i c l e

i n f o

Article history: Received 23 May 2016 Received in revised form 13 December 2016 Accepted 21 December 2016 Keywords: Indian cauliflower Microspore embryogenesis Genotypes Cold treatments Tetraploid FCM

a b s t r a c t Cauliflower (Brassica oleracea var. botrytis L.) is an important vegetable grown throughout the world. In India, wide diversity exists in cauliflower in terms of their adaptation to different temperature and maturity duration. However, there is no report regarding the microspore embryogenesis in different Indian cauliflower. Efficient microspore embryogenesis was optimized in all groups of cauliflowers with selection of genotypes and cold pre-treatments. One model genotype in each group has been identified for their wider application. Among the 30, 13 genotypes responded to microspore embryogenesis. Cold pre-treatment was found to be genotype specific. In late maturity genotype, Kt-119 microspore embryogenesis improved with 24–48 h of cold treatment. Flow cytometry analysis revealed more than 50% of the microspore derived plants as spontaneous diploids thus can be used directly as DH lines. Significant numbers of the microspore derived plants were haploids (15.77%) and tetraploids (17.07%). Colchicines treatment of 150 mg/l for a period of 36 h was most effective for chromosome doubling of the haploid plants with 73.3% of diplodization. Morphological and floral characterization revealed possibility of direct use of tetraploids in breeding programme as inbred line or as a parent to develop F1 hybrids with higher economic yield. The harvest indexes of the tetraploids were at par with the diploid and DH lines with normal male and female fertility. Development of triploid F1 hybrids using tetraploid line could be an alternative to the conventional hybrid breeding of cauliflower because of limited heterosis. Simple sequence repeats were used to genotype the DH and tetraploids generated from a hybrid along with their diploid parental lines. The microsatellite based markers produced only homozygous allele in DH and tetraploid lines. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Twenty million tonnes of cauliflower and broccoli are produced worldwide every year from 1.6 million hectares. China and India are the biggest producers at 8.9 and 6.7 million tonnes/year respectively – representing 74% of world production (www.yara. co.uk/crop-nutrition/crops/vegetable-brassica/key-facts/worldproduction/). Indian cauliflowers are very diverse in nature and typically adapted to high temperature and humidity because of their evolution under India condition in the last 200 years (Swarup and Chatterjee, 1972; Kalia et al., 2016). High degree of cross pollination and strong S- allele results in enormous heterozygosity. Thus, development of homozygous inbreds through conventional selfing

∗ Corresponding author. E-mail address: [email protected] (R. Bhatia). http://dx.doi.org/10.1016/j.scienta.2016.12.020 0304-4238/© 2016 Elsevier B.V. All rights reserved.

is practically impossible (Nieuwhof, 1963). Thus, development of complete homozygous lines through microspore embryogenesis and chromosome doubling will facilitate the improvement programme in great way. Another group of cauliflower namely snowball (Erfurt) types are cultivated widely throughout the world during winter season. This group of cauliflower is characterized by significant amount of self-pollination and very weak S allele. Therefore they have very narrow genetic base and development of heterotic hybrids is a challenge (Nieuwhof, 1963; Sharma et al., 2004; Dey et al., 2011). Development of tetraploid lines with higher economic yield and tetraploid based hybrids could be an alternative approach to overcome the less heterosis in this group of cauliflower. Application of haploids and doubled haploids in Brassicas has been discussed in detail by Ferrie and Möllers (2011). Isolated microspore culture is the most prominent methods for rapid development of DHs in all the crops. Besides its application in breeding

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programme and development of mapping population, DHs are immensely useful in studies related to genetic engineering, precise selection of desirable events through mutation and research related to metabolic changes in plants (Liu et al., 2005; Brew˙ Appiah et al., 2013; Zur et al., 2014). B. oleracea var. botrytis is being more recalcitrant to microspore embryogenesis as compared to the oil seed Brassicas like B. napus, B. juncea, B. catinata and B. rapa (Winarto and Teixeira da Silva, 2011; Gu et al., 2014). During the past two decades, microspore embryogenesis in B. oleracea has been reported by different workers with varying degree of success (Lemonnier-Le Penhuizic et al., 2001; Zhang et al., 2008; Winarto and Teixeira da Silva, 2011; Gu et al., 2014). However, development of protocols for highly efficient microspore embryogenesis is lacking in Brassica vegetables. Recently successful protocols for microspore embryogenesis in B. oleracea var. botrytis were developed in loose head cauliflower of China (Gu et al., 2014) and Indonesian cauliflowers (Winarto and Teixeira da Silva, 2011). Successful microspore embryogenesis depends on optimization of different factors for a particular genotype as in most cases genotype specific response is predominant. Factors like, genotypes, pre-treatments with high or low temperature, donor plant growing condition, developmental stage of the buds and media composition play very important role in successful microspore embryogenesis of different Brassicas, including B. oleracea (Takahata and Keller, 1991; Guo and Pulli, 1996; Tian et al., 2004; Chanana et al., 2005; Ali et al., 2008; Prem et al., 2008; Wang et al., 2009; Ferrie and Caswell, 2011; Winarto and Teixeira da Silva, 2011; Gu et al., 2014). Gu et al. (2014) has observed significant role of loose curd cauliflower genotypes cultivated in China in microspore embryogenesis. Recently, Winarto and Teixeira da Silva (2011) has examined the effects of factors like genotype, bud size, heat treatments and bud developmental stage in microspore embryogenesis in Indonesian cauliflower. Among the several factors, bud developmental stage was found to be most critical, however, donor genotypes play very significant role in the entire process. The genotypes and culture media content had significant role in microspore derived embryo formation in Chinese cabbage cultivar Lastochka (Shumilina et al., 2015). They have observed genotype dependent embryo formation in Chinese cabbage. Tuncer and Yanmaz (2011) reported the effects of high temperature and gamma irradiation shock treatments in microspore embryogenesis of B. oleracea var. acephala. Incubation temperature was also found to play important role in microscope culture of B. oleracea var. italica (da Silva Dias, 1999). In the present study, we have examined the effects of genotypes and cold pre-treatment in microspore embryogenesis of Indian cauliflower (B. oleracea car. botrytis). Later on flow cytometry was performed to analyze the ploidy level of regenerated plants. The main objective of the present study was to optimize different factors and to evaluate the usefulness of cauliflower lines with different ploidy level derived from microspore embryogenesis in future breeding programme. The possible use of higher ploidy level like tetraploids as inbred and parental line in hybrid breeding was also examined.

2. Materials and methods 2.1. Donor plants and growing condition The genotypes under study were raised in research field and glasshouse with semi-environment control facilities at ICAR-Indian Agricultural Research Institute, Regional Station, Katrain, Kullu, Himachal Pradesh, India. Four genotypes namely, Pusa Kartik Shankar, Pusa Sharad, Kt-34 and Kt-119 were used to study the effect of cold pretreatments on microspore embryogenesis. These genotypes represented cultivars from all the four maturity groups

of Indian cauliflower along with snowball types. The genotype Pusa Kartik Shankar was from the earliest group of cauliflower matures in the month of September. This group of cauliflowers is typically adapted to higher temperature and higher humidity. Pusa Sharad was from October maturity group. The first two groups of cauliflowers have yellowish curd with higher concentration of glucosinolates and other S-compounds. Therefore, they possess a distinct pungent flavor. One genotype, Kt-34 represents November maturity group with white compact curd. The genotypes of this group are closer to the Snowball/Erfurt type with white compact curd. One genotype, Kt-119 was from Snowball group grown during winter in the entire Northern plain and almost throughout the year in higher altitude of Himalayan region. The plants for microspore culture were raised as per the methods described in our earlier study (Bhatia et al., 2016) 2.2. Microspore isolation and culture The microspore isolation work was done between March-June as this period offers the most suitable weather condition for cauliflower curd development and flowering. For conducting the experiment flower buds in the late uninucleate to early binucleate stage were harvested from the cauliflower plants. The flower buds were collected from the raceme when first 1–3 flowers opened. The buds with normal development were selected for this purpose. The ratio of petal and anther length (P/A: 1–1.2) was used to determine the normal development of the buds (Gu et al., 2014). The method described by Cousin and Nelson (2009) in B. napus was followed to prepare the microspore suspension with minor modification. The microspore density was adjusted to 4 × 104 microspores/ml determined using Neubauer haemocytometer counting chamber (Bioanalytic GmbH, Germany). Microspore suspension (10 ml) was dispensed into 90 mm sterile Petri dish (Tarsons, India). Thereafter activated charcoal (0.01%) was also added to each plate. Petri dishes were organized as per the replications and treatments. Petri dishes were sealed with Parafilm and cultures were incubated at 32.5 ◦ C (dark) for two days. The experiments were laid out in completely randomized block design with three replications and three petri plates per replication. 2.3. Cold pre-treatment Four genotypes from different maturity groups of cauliflower were taken for the cold treatment. The buds with the highest percentage of late uni-nucleate to early bi-nucleate stage were collected. The bud sizes with highest percentage of microspore in the desirable stage were standardized in another study using fluorescent microscopic methods (Winarto and Teixeira da Silva et al., 2011). The genotypes, Pusa Kartik Shankar and Pusa Sharad had highest percentage of late uni-nucleate and early bi-nucleate microspores in the size 4.0–4.5 mm. While the genotypes Kt-34 and Kt-119 with bud size of 4.5–5.0 mm had highest percentage of microspore at desirable developmental stages. After surface sterilization for 10 min in 0.1% (w/v) mercuric chloride, the buds were then placed into Petri dishes containing cold (4.0 ± 1.0 ◦ C) NLN13 medium. The tightly sealed petri dishes with double layers of Parafilm were placed for 0.0–3.0 days at 4.0 ± 0.5 ◦ C in the laboratory refrigerator. Microspore isolation and culture procedure was carried out as described above. After 40–45 days of culture, the number of cotyledonary embryos that were longer than 0.5 mm were counted. The experiment was laid out in completely randomized block design using three replications with three Petri plates per replication. All data were subjected to standard analysis of variance (ANOVA) to test for significance among different treatments. Duncan’s multiple range test was used to detect significant differences among the mean values of traits.

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2.4. Microspore embryogenesis and plant regeneration When the white translucent embryos were visible to the naked eyes, the plates were transferred to an incubator shaker in dark with 25.0 ± 01 ◦ C temperature at 50 rpm shaking. After 40–45 days of microspore isolation, the embryos at cotyledonary stage were counted. Microspore derived embryos measuring 4–6 mm in length were transferred on to embryo regeneration medium {(1.0X MS medium (Murashige and Skoogs, 1962) supplemented with 1.0 mg l−1 6-benzylaminopurine (BAP), 1.0 mg l−1 , Zeatin, 0.2 mg l−1 , 2% sucrose, (pH. 5.75) and 0.7% agar (Merck, Mumbai, India)} for germination (Bhatia et al., 2016). The regenerated embryos were transferred to MS medium with 2.5 mg l−1 Thidiazuron (TDZ), 1.0 mg l−1 6-furfurylaminopurine (Kinetin), 0.2 mg l−1 Naphthalene acetic acid (NAA; Duchefa, Netherland) and 0.1 mg l−1 Gibberellic Acid (GA3 ; Duchefa Biochemie, Netherland), 2% sucrose and 0.8% agar (pH. 5.8) for multiplication (Bhatia et al., 2016). The microspore embryo derived shoots measuring 3.5 cm in length were rooted on half strength liquid MS medium with 45 g l−1 sucrose and 1.5 mg l−1 Indole-3-Butyric Acid (IBA: Duchefa Biochemie, Netherland) (Bhatia et al., 2015). The microspore embryos were maintained at 25 ± 1 ◦ C under fluorescent white light (47 ␮mol m−2 s−1 ) at a photoperiod of 16:8 h light and dark cycle for embryo germination and rooting. The rooted plants were gradually acclimatized in polythene bags containing sterile cocopeat: perlite and vermiculite mixture (1:1:1) as per the standard practice of our station. They were placed in naturally ventilated glasshouse for acclimatization (max temperature: 25◦ C; min temperature: 15◦ C). After 2–3 weeks of acclimatization, the growing plants were transferred to 12 in. plastic pots with same potting mixture. 2.5. Ploidy determination through flow cytometry analysis The flow cytometry analysis of the samples for ploidy determination was done as per the protocol described by Bhatia et al. (2016). The stained nuclei samples were analyzed using a BD FACS Canto II (BD Biosciences, San Jose, CA) flow cytometer with a 488nm laser and fitted with a high throughput sampler (HTS). The FCM work was carried out at CSIR-Indian Institute of Himalayan Bio resource Technology, Palampur, Himachal Pradesh, India. The diploid cauliflower variety Pusa Snowball K-1 was used as control diploid. The ploidy level of microspore derived plants were determined by comparing the DNA content of the microspore derived plants at G0 and G1 stage with the diploid genotype Pusa Snowball K-1. 2.6. Doubling of chromosome number of the haploids The identified haploid plants from microspore-derived embryos were treated with colchicine for diplodization and development of DH lines. The plants were initially transferred to plug tray with a potting mixture of cocopeat: vermiculite: perlite (3:1:1) after rooting under in vitro condition. Once the ploidy number of the microspore derived plants was determined through FCM, the haploids plants were treated with colchicine. For optimizing the colchicines treatment all the haploid plants of different genotypes were taken. They were treated with five different concentration of colchicine (100 mg l−1 , 125 mg l−1 , 150 mg l−1 , 175 mg l−1 and 200 mg l−1 ) for five different duration (12 h, 24 h, 36 h, 48 h and 60 h). Colchicine was applied through root dip treatment. For each treatment, five plants per replication were used and each treatment was replicated thrice. The plants were taken out from the tray and roots were washed with distilled water and dried properly before the treatment. The plants were again checked for ploidy level after colchicines treatment. After optimizing the colchicine treatment

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rest of the haploid plants from different genotypes were treated with colchicine. However, ploidy level was confirmed through FCM before use of the plants in further study. The colchicines concentration and duration of treatment were taken as two factors in interaction study using the COSTAT for windows at 5% level of probability. 2.7. Morphological and floral characterization of plants with different ploidy level One F1 hybrid Pusa Snowball K-1 x Kt-34 was developed by crossing two responsive genotypes Pusa Snowball K-1 and Kt34. Two hundred microspore dervied plants from the hybrid Pusa Snowball K-1 x Kt-34 were characterized for important morphological traits viz. i) Plant vigor ii) plant height (cm) iii) leaf length (cm) iv)leaf width (cm) v) gross plant weight (Kg) vi) marketable curd weight (Kg) vii) net curd weight (Kg) viii) core length (cm) ix) curd appearance x) curd color xi) curd compactness xii) harvest index (%) xiii) pollen viability xiv) petal length (mm) and xv) petal width (mm). For each trait these two hundred genotypes were grouped into 3 different categories. These morphological data were later compared with the flow cytometer data to study agronomic and other traits of the plants with different ploidy level. Curd appearance was recorded on visual observation on curd development. Compactness was determined by thumb pressing of middle of the curd 20 days after curd initiation. Curds were classified intro four different groups based on compactness (Very compact, compact, medium compact and loose). Harvest index was calculated by dividing net curd weight with gross plant weight. Harvest index is the measure of capability of any particular genotype to convert the total biomass towards the development of economic plant part. Pollen viability was measured by pollen stainability test. Freshly opened flowers were collected and pollen grains were released from anthers on microscopic slide. Pollen viability was assessed with 1% aceto-carmine solution. The numbers of the stained pollen were observed under a compound microscope. Three hundred pollen grains were counted for each plant. For morphological and floral characterization, five replications with five plants per replication were used for each genotype. All data were subjected to standard analysis of variance (ANOVA) to test for significance among different treatments. Duncan’s multiple range test was used to detect significant differences among the mean values of traits. 2.8. Genotyping of DH and tetraploid population using the SSRs Leaf tissue was collected from parental lines of the hybrid, Pusa Snowball K-1 x Kt-35 along with the DH and tetraploid population developed from this hybrid. DNA was extracted from leaf tissue using a modified CTAB method. Seven pairs of SSRs were identified among 83 B. oleracea SSRs across the genomes (Wang et al., 2012) based on their polymorphic amplification among these two parental lines. The DH and tetraploid plants were genotyped using these seven pair of microsatellite markers. For determining the genetic fidelity of the DH and tetraploid plants, ten plants each derived from 3 plantlet of DH and tetraploids were also genotyped using 30 SSRs. Additionally 40 RAPD were used to confirm genetic fidelity. 3. Results 3.1. Influence of genotypes in microspore embryogenesis In this study, 30 genotypes from different maturity groups were assessed for their response to microspore embryogenesis. Thirteen genotypes responded to microspore embryogenesis with varied number of embryos (Table 1). Two genotypes from the late

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Table 1 List of Indian and snowball cauliflower genotypes used for microspore culture. Sl. No.

Genotype

Maturity group

Temperature requirement for curd formation and development

Salient features

Mean embryo yield (Embryos/petri dishes)

Highest embryo yield (Embryos/petri dishes)

Mean rate of germinated embryo (%)

1 2

Pusa Meghna Pusa Kartik Shankar (F1 hybrid) Kt-50 Kt-60 Kt-103 Kt-113 Kt-139 Pusa Sharad Pusa Shukti Kt-87 Kt-58 Kt-65 Kt-118 Kt-117 Kt-115 Kt-114 Kt-107 Kt-221 Pusa Pausija Pusa Himjyoti Kt-32 Kt-31 Kt-33 Kt-34 Pusa Snowball K-25 Pusa Snowball K-1 Kt-119 Kt-27 Kt-19 Pusa Snowball-1

September September

20–27 ◦ C 20–27 ◦ C

Indian type with compact curd Indian type with compact curd

0 ± 0.0c 17.1 ± 6.9b

0 38.3

– 43.8

September September September September September October October October October October October October October November November November November November November November November November December onwards

20–27 ◦ C 20–27 ◦ C 20–27 ◦ C 20–27 ◦ C 20–27 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 20–25 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 16–20 ◦ C 10–16 ◦ C

Indian type with loose curd Indian type with loose curd Indian type with loose curd Indian type with loose curd Indian type with loose curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with medium compact curd Indian type with compact curd Indian type with compact curd Indian type with compact curd Indian type with compact curd Snowball type with compact white curd Snowball type with compact white curd Snowball type with compact white curd

0.7 ± 0.6c 0 ± 0.0c 0 ± 0.0c 1.2 ± 0.7c 0 ± 0.0c 15.6 ± 5.6b 0.8 ± 0.7c 0 ± 0.0c 0 ± 0.0c 0 ± 0.0c 0.8 ± 0.4c 0 ± 0.0c 0 ± 0.0c 0 ± 0.0c 2.6 ± 1.4c 0 ± 0.0c 0 ± 0.0c 1.3 ± 0.7c 0 ± 0.0c 0 ± 0.0c 0 ± 0.0c 16.9 ± 4.8b 13.5 ± 5.3b

2.5 0 0 3.6 0 54.0 3.7 0 0 0 2.1 0 0 0 4.6 0 0 6.3 0 0 0 59.2 65.2

37.6 – – 45.2 – 42.7 25.6 – – – 28.8 – – – 42.3 – – 82.4 – – – 73.5 29.5

December onwards December onwards December onwards December onwards December onwards

10–16 ◦ C 10–16 ◦ C 10–16 ◦ C 10–16 ◦ C 10–16 ◦ C

Snowball type with compact white curd Snowball type with compact white curd Snowball type with compact white curd Snowball type with compact white curd Snowball type with compact white curd

28.5 ± 6.8a 30.2 ± 6.4a 0 ± 0.0c 0 ± 0.0c 13.5 ± 5.6b

84.5 88.9 0 0 43.7

33.7 64.2 – – 38.2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Same letter in the column represents no significant different at 5% level of probability.

maturity groups (snowball type), Kt-119 and Pusa Snowball K-1 produced highest number of embryos per petri dish. The genotypes from late maturity groups were more responsive to microspore embryogenesis as compared to the early and mid maturity genotypes. Among the 10 genotypes from September maturity group, only three genotypes namely Pusa Kartik Shankar, Kt-113 and Kt-50 responded to microspore embryogenesis and rest of the 7 genotypes did not produce any microspore embryos. The various stages of microspore embryogenesis are presented in Fig. 1. Highest numbers of embryos were produced in the genotype, Pusa Kartik Shankar (17.1 embryos/petri dish) followed by Kt113 (1.2 embryos/petri dish) and Kt-50 (0.7 embryos/petri dish). Three among the 8 October maturity genotypes viz. Pusa Sharad (15.6 embryos/petri dish), Pusa Shukti (0.8 embryos/petri dish) and Kt-118 (0.8 embryos/petri dish) responded to microspore embryogenesis. In November maturity, 3 among 9 genotypes had successful embryogenesis. Kt-34 had highest number of embryos per perti dish (16.9) while, Kt-107 and Pusa Himjyoti had very low number of embryos. Among the snowball genotypes, 4 among 6 produced microspore embryos and each of them had more than 10 embryos/petri dish. Among the snowball types, highest number of embryos were produced in the genotype Kt-119 followed by Pusa Snowball K-25, Pusa Snowball K-1 and Pusa Snowball-1 (Fig. 2). 3.2. Effects of cold treatment on microspore embryogenesis Four genotypes (Pusa Kartik Shankar, Pusa Sharad, Kt-34 and Kt-119) representing four different maturity groups were used to determine the influence of cold treatments on microspore embryogenesis. The flower buds of different genotypes were collected when they had highest number of microspores in late uninucleate

Table 2 Effect of cold pre-treatment on microspore embryogenesis in cauliflower. Donor genotype

Cold pretreatment (h)

No. of embryo yield per flower bud ± SE

Pusa Kartik Shankar

0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72

20.6 ± 3.4 a 13.1 ± 1.8 b 5.0 ± 1.2 c 1.7 ± 0.4 c 21.5 ± 2.8 a 16.9 ± 2.3 b 13.2 ± 1.4 b 1.8 ± 0.3 c 24.7 ± 2.9 a 25.4 ± 2.6 a 26.7 ± 2.9 a 6.2 ± 1.9 b 32.0 ± 1.3 b 42.8 ± 1.7 a 38.7 ± 2.5 a 3.4 ± 1.2 c

Pusa Sharad

Kt-34

Kt-119

Same letter in the column represents no significant different at 5% level of probability.

to early bi-nucleate stage. Bud size of 4.0–4.5 mm for the genotypes Pusa Kartik Shankar and Pusa Sharad and 4.5–5.0 mm for the genotypes Kt-34 and Kt-119 had highest number of microspores at desirable developmental stage. The flower buds of all the four genotypes when treated for 24–72 h had different response to cold treatments (Table 2). Two early maturity genotypes, Pusa Kartik Shankar and Pusa Sharad had highest number of embryos in control without any cold treatments. Microspore embryo yield was reduced significantly in

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Fig. 1. The different stages of Brassica oleracea microspore cultures. a–b Isolated microspores, after induction and embryogenic microspore. The embryogenic multi-nucleate microspores are indicated by arrow. c–d different stages of microspore embryos as visible 15 days after microspore isolation under phase contrast inverted microscope (c) and stereozoom microscope (d); e Heart-shaped embryo. f Cotyledonary embryo. Bars in a–b: 50 ␮m; c: 120 ␮m; d: 300 ␮m; e–f: 100 ␮m.

both the genotypes with increasing duration of cold treatments. The genotypes with low temperature requirement and mature late responded to cold treatment. The genotype Kt-119 produced highest number of embryos when treated for 24 h. Cold treatment for 24 h or 48 h produced significantly more number of embryos as compared to the control. However, increasing the duration to 72 h significantly reduced the embryo yield as compared to the control and other treatments. The genotype Kt-34 had same number of embryos in control, cold treatment for 24 h and cold treatment for 48 h. However, embryo yield was reduced significantly when treated for 72 h. 3.3. FCM to determine ploidy level of the microspore plants The ploidy level of the regenerated plants was checked by FCM ploidy analyzer before their planting in the field. Agronomic traits of the plants were also checked in vegetative and flowering stage. Total 956 plants from 8 genotypes were used for ploidy level analysis. These genotypes represent cauliflower from different maturity groups. Rest of the microspore derived plants was not used in the present FCM study. Cauliflower cultivar Pusa Snowball K-1 was used as a standard diploid genotype. The ploidy of the microspore

derived plants were determined by comparing the cell division activities with the control diploid genotype (Figs. 3 and 4). Among the analyzed samples, 15.77% of the plants were haploids, 53.21% of the plants were spontaneous doubled haploids and 17.07% were tetraploids. The frequency of aneuploids and chimeras were 9.0%. Among the different genotypes, highest percentage of spontaneous doubled haploids were recorded in the genotype, Pusa Snowball K-25 (62.6%) followed by Pusa Sharad (57.9%) and Pusa Shukti (57.3%). The minimum percentage of spontaneous doubled haploids were observed in Pusa Snowball K-1(39.6%). Pusa Shukti, Kt-119 and Pusa Snowball K-1 x Kt-33 had more than 20% haploids. Highest frequency of tetraploids was found in the Pusa Kartik Shankar derived population followed by Kt-34 and Pusa Snowball K-1 populations. The frequency of triploids pentaploids and other ploidy level was less than 5% (Table 3). 3.4. Diplodization of haploid plants through colchicines treatment Among the five different concentrations of colchicine, a concentration of 150 mg/l with duration of 36 h was most effective for root dip treatment. This treatment produced 73.3% normal diploids plants (DHs) from the haploids. Colchicine concentration of

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Fig. 2. Response of the 4 different genotypes to microspore embryogenesis under the optimized condition standardized for different groups of cauliflower a very poor microspore embryogenesis in the genotype Kt-113 b microspore emnbryogenesis in Kt-107 c moderate numbers of microspore embryos in the genotype Pusa Snowball K-25 d highest microspore embryogenesis in the genotype Kt-119 in the present study. 1 bar = 90 mm.

Table 3 FCM Ploidy level in microspore-derived plantlets of different groups of cauliflower cultivated in India. Table Genotype

No. of plantlets tested

Haploid (%)

Diploid (%)

Triploid (%)

Tetraploid (%)

Pentaploids/ mixoploids

Aneuploid and chimera (%)

Pusa Kartik Shankar (F1 hybrid) Pusa Sharad. Pusa Shukti Pusa Himjyoti Kt-34 Pusa snowball K-1 Pusa Snowball K-25 Kt-119 Pusa Snowball K–1 x Kt-33 Average

115 87 51 76 134 102 119 76 311 956

11.7 10.4 23.4 13.2 12.7 14.7 12.6 23.1 20.1 15.77

51.8 57.9 57.3 54.9 52.4 39.6 62.6 52.6 49.8 53.21

3.2 2.5 1.9 2.7 2.6 0 0 5.9 3.7 2.50

27.4 19.4 13.2 12.8 23.5 24.7 16.2 3.6 12.8 17.07

3.5 3.3 2.9 5.6 0 7.4 0 0 0 2.52

3.4 6.5 1.3 10.7 8.8 13.5 8.6 14.7 13.5 9.00

Table 4 Effects of colchicines concentration and duration of treatment for diplodizing the haploid plants. Colchicine concentration

100 mg/l 125 mg/l 150 mg/l 175 mg/l 200 mg/l

Duration of colchicines treatment 12 h

24 h

36 h

48 h

60 h

10.0 a 10.0 a 30.0 a 16.7 a 13.3 a

20.0 a 23.3 ab 53.3 bc 36.7 bc 11.0 c

33.3 a 43.3 ab 73.3 bc 56.7 c 26.7 c

13.3 a 16.7 a 26.7 a 23.3 a 30.0 a

36.7 a 16.7 a 36.7 a 26.7 a 23.3 a

Same letter in the column represents no significant different at 5% level of probability.

175 mg/l and 150 mg/l for 36 and 24 h, respectively produced 56.7% and 53.3% DH plants from the haploids. When the colchicine concentration increased beyond 150 mg/l the diplodization frequency decreased in all the cases (Table 4). When the concentration was above 150 mg/l, instead of diploids other ploidy level like triploids and tetraploids produced in larger frequency. In a concentration

of 200 mg/l mortality of plants was also significant (data not presented here). In studying the effects of these two factors both the factors were had significant role individually’ however, the interaction of concentration x period of treatment was found to be non-significant.

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Fig. 3. FCM analysis of the microspore derived plants to determine ploidy level a FCM of the control diploid plant sample of the cauliflower cultivar Pusa Snowball K-1 b FCM of the haploid plants c FCM analysis of the tetraploid plants d FCM of one aneuploid microspore derived plants with probale chromosome elimination from the haploid genome.

ball K-1 x Kt-35 were characterized for various agronomic and floral traits along with their parental lines (Table 5). All the haploid plants were male sterile with shrivelled anther and non viable pollen grains. The DH plants have around 80% pollen viability which was at par with the parental lines Pusa Snowball K-1 and Kt-35. Interestingly, the tetraploid plants have also normal pollen viability (76.9%). The plants were very vigorous in the tetraploid lines with much larger leaves. Curd size was also significantly high as compared with the diploid parental lines and DHs in the tetraploids. The haploids plants were very weak and in several cases did not form any marketable curds. Almost 20% of the haploids have broccoli like very small flower head. However, the agronomic and floral data were collected from the haploids with curd like structures. The curd size was very small as compared to the normal diploids and tetraploids (Fig. 5).

3.6. SSR genotyping of the DH and tetraploid plants Fig. 4. Bar diagram depicting the percentage of plants with different level of ploidy determined through FCM analysis of microspore derived plants of cauliflowers.

3.5. Characterization of microspore derived plants Ten randomly selected plants of three ploidy levels (haploids, diploids and tetraploids) generated from the hybrid, Pusa Snow-

Seven sets of SSRs identified among 89 pairs of B. oleracea SSRs were found to produce polymorphic amplification in the parental lines of the hybrid, Pusa Snowball K-1 x Kt-35 were taken for the molecular analysis. All the DH and tetraploids plants produced only homozygous maternal or paternal allele. All the plants derived from

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Table 5 Morphological characterization of the microspore derived plants. Sl. No.

Traits

Pusa Snowball K-1

Kt-33

DH

Haploids

Tetraploids

LSD 0.5

1 2 3 4 5 6

Normal 51.55 ± 4.5 b 48.31 ± 4.0b 24.45 ± 2.76ab 2.83 ± 0.20b 1.55 ± 0.19b

Normal 39.71 ± 4.47d 34.75 ± 3.53d 20.18 ± 2.57c 2.56 ± 0.25b 1.34 ± 0.22b

Normal 46.80 ± 4.57c 43.09 ± 5.36c 22.34 ± 2.70bc 2.64 ± 0.41b 1.37 ± 0.21b

Weak 27.61 ± 4.50e 23.96 ± 4.05e 13.08 ± 2.23d 1.28 ± 0.34c 0.61 ± 0.14c

Very vigorous 64.50 ± 6.91a 59.18 ± 7.68a 25.52 ± 2.88a 3.30 ± 0.49a 2.11 ± 0.29a

4.58 4.64 2.37 0.32 0.19

7 8 9

Plant vigor Plant height Leaf length Leaf width Gross plant weight Marketable curd weight Net curd weight Core length Curd appearance

1.05 ± 0.18a 6.55 ± 0.28a Normal

0.95 ± 0.14a 5.69 ± 0.33b Normal

Curd color Curd compactness

Snow white Vey compact

White Very compact

1.14 ± 0.29a 6.75 ± 0.51a Larger curd with normal development White to cream Very compact

12 12 13 14

Harvest index Pollen viability Petal length Petal width

37.20 ± 5.57a 80.20 ± 7.67a 19.09 ± 1.76ab 8.58 ± 0.61a

37.30 ± 6.17a 77.50 ± 9.70a 18.32 ± 1.21b 8.44 ± 0.90a

36.16 ± 8.75a 83.10 ± 8.49a 19.89 ± 1.22ab 8.43 ± 0.88a

0.26 ± 0.08b 4.12 ± 0.82c Poorly developed and loose and ricey curds in most cases Yellowish white Medium compact to loose 21.02 ± 7.10b 0.00 ± 0.00b 13.36 ± 1.30c 5.40 ± 0.60b

0.17 0.48

10 11

0.94 ± 0.20a 6.20 ± 0.58a Normal but 2 plants with ricey curd White Very compact

34.81 ± 8.82a 76.90 ± 9.77a 20.31 ± 2.03a 8.35 ± 1.03a

6.66 7.21 1.38 0.74

Same letter in the row represents no significant different at 5% level of probability.

the single DH and tetraploids produced similar bands they were also confirmed with 10 RAPD markers. 4. Discussion In Indian cauliflower, we are reporting for the first time the influence of cold treatment, various media composition and ploidy analysis for rapid development of doubled haploid population. Possibility of using the higher ploidy level like tetraploid in breeding programme for increasing productivity has also been examined. Microspore embryogenesis in different crops is highly sensitive to several factors. Growing environment of donor plant is one of the major factors determining the ability of the microspore to pass through the sporophytic phase. Genomic composition and architecture of the donor plants is considered to be the most critical factor for successful microspore embryogenesis. Thus information regarding the response of any particular genotype is prerequisite before starting large scale DH based breeding programme and genetic analysis. Microspore culture response varies among genotypes within a species. Ferrie and Caswell (2011) have reviewed extensively the role of genotypes in successful microspore embryogenesis. Genotypic differences were apparent when 15 out of 19 genotypes of B. oleracea formed embryos and embryo yield ranged from 0 to 3000 per 100 buds (Ferrie et al., 1999). Recently Gu et al. (2014) reported 14 among 17 genotypes of loose and tight head cauliflower produced microspore embryos, however, embryo yield per flower bud varied between 0 and 58.2. In our study, more than 50% of the genotypes (17 among 30) did not respond to microspore embryogenesis. Among the various maturity groups, the snow ball type (late) cauliflower genotypes were more favorable to microspore embryogenesis. Winarto and Teixeira da Silva (2011) reported that B. oleracea var. botrytis cultivar had a significant effect on microspore embryogenesis. Among the different Indonesian cauliflowers, ‘Kemeh’ showed the highest embryogenic response, with up to 11 embryos/3 buds on average. This cultivar produced significantly more embryos—independent of bud size—than other cultivars. Recently, Shumilina et al. (2015) also reported the influence of genotypes in microspore embryogenesis of Chinese cabbage. They have reported a 10 fold difference in responsiveness among individual genotypes. Recent results on influence of genotypes in microspore embryogenesis of B. oleracea (ornamental kale) is being reported by (Wei et al., 2008). Microspore culture response varies among genotypes within a species, as is commonly found with tissue culture techniques.

Cold pretreatment in microspore culture has been successfully employed in broccoli (Yuan et al., 2011), and appears to be useful in recalcitrant B. oleracea microspore culture. As there was no previous study in Indian cauliflower we are reporting 4 model genotypes for microspore embryogenesis one each from different maturity groups for their wider use in genetic studies and application in breeding programme. Recently, a study by Gu et al. (2014) in loose curd cauliflower indicated that the cold pretreatment effect was genotype-dependent. Among the three genotypes studied by Gu et al., 2014; 1–4 days of cold pretreatment efficiently induced microspore embryogenesis to different degrees. This study concluded that cold pretreatment effect was genotype-dependent, but the application of cold pretreatment could effectively produce embryos because collection and sterilization of flower buds was a one-time event, saving time and labor. In our study it was evident that the genotype from the late maturity group responded positively to cold pre-treatment for 24–48 h. In rest of the three genotypes from 3 different maturity groups cold pre-treatment had adverse effect on microspore embryogenesis. Cold treatment had therefore adverse effect on all the genotypes of Indian cauliflowers Therefore, before applying any treatment the suitability in a specific genotype need to be optimized. Characterization of microspore derived plants for their ploidy level is vital for utilization of these plants in genetic studies and breeding programme. Development of quick and reliable method for ploidy determination is vital for large scale use of this technique. Chromosome doubling of the haploids developed through isolated microspore culture is needed for further use of the plants practical breeding programme. Spontaneous chromosome doubling of microspore derived embryos is a common phenomenon in large number of plant species including Brassicas. Gu et al. (2014) reported a large percentage of B. oleracea microspore derived plants were diplodised spontaneously in the culture medium. In our study we have also observed more than 50% of the microspore derived plants were spontaneous diploids. However, percentage of plants with different ploidy level varied among genotypes. Spontaneous diploids were as high as around 75% to below 50%. Most of the earlier studies reported high number of haploid plants in different Brassica species (Duijs et al., 1992; da Silva Dias, 1999; Gu et al., 2004a; Takahira et al., 2011). The composition of culture medium and individual genotype play very important role in spontaneous diplodization of the microspore derived plants. However, more understanding is needed to determine the role of different

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Fig. 5. Characterization of haploid,diploid and tetraploid plants derived from the F1 hybrid, Pusa Snowball K-1 × Kt-34 for various agronomic and floral traits. a Leafs of haploid, diploid and treaploid plant during the stage of curd initiation b and c curds of haploid, diploid and tetraploid line 20 days after curd initiation d floral characteristics of haploid, diploid and tetraploid plant e flowers of haploid, diploid and tetraploid plant with separated sepals and petals. It depicts the normal male and female fertility in the diploid and tetraploid plant and male sterile shriveled anther in the haploid plant. f siliquas of the diploid and tetraplants developed theough bud pollination 15 days after pollination, in haploid plants no seed setting was possible because of non viable pollen grains. T: Tetraploid, D: Diploid, H: Haploid.

factors in changing the ploidy level of haploids microspore derived embryos. There was significant number of tetraploids along with the diploids produced spontaneously without any treatment with chromosome diplodizing agent. Very interesting results were recorded while characterizing haploids, diploids and tetraploids along with the parental lines of the donor hybrid for various agronomic and floral traits. It is well known that doubling in ploidy level increase the general vigor of plants and used as a breeding method in few vegetable crops with leaf as economic part (e.g. Indian spinach). However, application of tetraploids in normal diploid crops is limited. We are reporting for the first time that in cauliflower tetraploids could be very useful as they have all desirable characters of cauliflower. The tetraploid lines had more than 50% economic yield as compared to the diploids. They have normal fertility as pollen viability was at par with the normal diploids and

DH lines. We succeeded to produce triploid hybrid by using these tetraploid lines as pollen parent with diploid CMS line as female. It was found that these hybrids have very good vigor with good curd characters for their use in commercial scale (data not presented here). In cauliflower in general and snowball cauliflower in particular heterosis is very low because of narrow genetic pool (Newhof, 1963). Thus, polyploidy based breeding strategy could be an alternative to the conventional breeding programme in cauliflower. However, chromosomal stability of the tetraploid lines needs to be worked out before their long term use.

Acknowledgement We are highly thankful to Science and Engineering Research Board, Department of Science and Technology, Govt. of India for financial assistance in carrying out this research work.

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