“Development of double haploids for production of bacterial wilt resistance and high yielding brinjal (Solanum melongena L.) lines by using anther culture Technique”
Loading...
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.)
Abstract
An investigation was carried out to “Development of double haploids for production of bacterial wilt resistance and high yielding brinjal (Solanum melongena L.) lines by using anther culture Technique at Department of Vegetable Science under All India Coordinated Project on Vegetable crops in collaboration with Richaria Research Laboratory, tissue culture laboratory of the Department of Plant Molecular Biology and Biotechnology, IGKV Raipur, under well sterilized and controlled condition in the Rabi during 2017-18 and 2018-19. There are five aspects were study viz. (i) Identification of ideal stage of flower bud, size anther size and microspore development stage.(ii) Standardization of pre-treatment duration, culture condition and media for inducing haploid callus through anther culture under this three sub aspects were discussed (a) Identification ideal flower bud, pollen stage for anther culture (iii) Optimization of effective invitro regeneration for production of haploid plants from anther derived Callus, (iv) Development of double haploid plant population with colchicine treatment in different brinjal lines (v) Hardening and confirmation of anther culture derived DH plants. Anthers containing pollen grains at the mid-late uninucleate-early binucleate stage in flower bud size of 12-15.9 mm ideal for antherculture having more vacuolated microspores and it depends on genotype (anthers had greenish yellow colour at 4.0-6.2mm).
Anthers containing pollen grains at the mid-late uninucleate-early binucleate stage in flower bud size of 12-15.9 mm were exposed at 5℃ for 1-3hr duration were before cultured on six media different C medium vize C0 C1, C2, C3, C4, C5, N6 treatments in combination with two different incubation temperatures 25oC for 3 weeks and 35oC for 8 days under dark condition. The maximum anther derived calli induction frequency obtained in treatment combination of cold pre-treatment 3h + C5 callus induction media + 25℃ heat stress for 21 days (74.05 %) with in moderate number of days 12.84 creamy white friable calli and next better treatment was 2h + C2 + 25℃ (64.39 %) C5 media requires moderate number of days with 3h pretreatment 15.02 days at 25℃ and 10.66 days at 35℃ heat stress. The treatment1h+N6 media + 25℃ required maximum of 85.17 days for callusing with poor calli production (0.89%).
The maximum shoot initiation as calluses transfer on T5e treatment that is MS medium containing NAA, Kin and BAP (0.2:1:3) mg/l had 82.17 % green calli within 23.83 days. The initiated shoots are then subsequently transferred to shoot elongation medium MS media fortified with GA3 0.2 mg/l in which multiple shooting occurs. The maximum rooting response had the plantlets cultured on MS media supplied with IBA-1mg/L (76.17%) and R2 -GR free MS media (68.23%).
The results showed significant in medium and genotype effects for the androgenic capacity of callus induction and regeneration. Almost all genotypes responded well in callus induction at 25℃ for 3 weeks darkness in C5 media IGB-17 × PS and IGB-17 × KT had highest calli frequency 76.29% and (74.05%), least calli production had in BRLVAR-1(29.11%) at 25℃ heat stress. Maximum size of calli recorded in same media in IGB-17 × PS (9.88 mm), IGB-54×PS (9.19 mm) and 9.10 mm in C4 in IGB-17. Earlier calli induction noticed in IGB-17 + C3 (8.79 days), IGB-17 × KT + C3 (8.90days), KS + C4 (9.47 days) and late in IGB-55 × PS + N6 (57.05 days). Among the genotypes IGB-54 × PS, IGB-17 × KT, IGB-17 × PS exhibited maximum mean calli frequency 35.59%, 34.89 % and 33.28% respectively. The anther derived calli cultured on regeneration media T5e the genotype IGB-54 × BWL (88.06%) had maximum greening frequency fallowed by IGB-17 × KT (84.75%), and IGB-54× PR (80.00%). But the highest shoot initiation (78.56%) and shooting response (51.73%) had in IGB-54× PR in T5e. Regeneration of both embryoid + calli was formed culture anther at 35 ℃ temperature stress in IGB-17 × KT (C5), BRLVAR-1(N6), IGB-54 × IBWL(C5) with embryoid formation frequency in C5 (2.00 %), N6 (1.33%) and C5 (0.65%) but the KS (C6 media) had direct plantlet formation (0.64%). In ten genotypes at 35℃ temperature for 8days stress condition, very few calli induced but poor in quantity and quality, regeneration of calli maximum Embryoid formation and plant frequency (%) observed in C1 media induced calli subcultured on T5a 5.34% and 7.33%, respectively in IGB-17 × KT. All the genotypes showed response to rooting in both R2 media IBA-1mg/l and R1 highest rooting had with IGB-17 × KT + 1mg/l IBA (76.17%).
Plantlets examined morphologically and cytologically compared with their diploid parental plants, haploid plants were found to be less size for leaf, stem diameter, growth vigour and flower size. Normally haploids were showed pale corolla, anther and stigma color, plant length and flower size were differ into less and equal, also flower color of donor. Haploid plants were exhibited slow growth; delayed flowering, less in number / plant, anther number /flower and the anther formed in the third layer were almost empty. The maximum haploid frequency of 92.59 % in IGB-17 fallowed by 83.05 per cent observed in IGB-17 × KT, 80.50 per cent in IGB-54 × KT, and 80.28 per cent in IGB-54 × PS. The cross IGB- 54 × PR had maximum survival of 60.18 %. Out of this 75% plants were treated with colchicine 0.02-0.1% at 4-6 leaf seedling stage by injection. Ploidy level was identified in each androgenic regenerants of each cross di haploid , viz.46(76.92%) dhs, 48 (76.19%) in IGB-54 × PR, 67.24% in IGB-54 × KT,65.00% in IGB-54 × PS, 58.34% in IGB-17 × KT,64.00 % in IGB-17,50.00% in IGB-55 × PS, 57.14 % in KS and 52.38% in BRLVAR-1.
The androgenic regenerants of each donor cross leaf, flower and fruit morphology evaluated individually. The androgenic derived dhs from IGB-54× IBWL, IGB-54× PR were showed maximum variability in corolla color, anther color and their number. IGB-17 × PS showed pale corolla colour and reduced size of inflorescence, while IGB-17 × KT had reduced plant height; petal size, pale petal color and greenish anther in reduced size were noticed. But in DH s of IGB-17and IGB-54 × PR fruit colour same as in the donors. Around 76.92 % DHs of IGB-54 × IBWL, 87.50 % plant in IGB-54 × PR had green leaf blade colour, remain15.38 % in IGB-54 × IBWL (4) slight dark green and 7.69 % VG. In IGB-17 , IGB-17 × KT, IGB-17 × PS, IGB-54 × PS dhs 71.43%, 73.47% , 70.00 %, 62.50% showed pale violet as observed in it donor crosses.
This Fr- S, Fr-Curve; Fr- under C exhibited no variability among the DH individuals in IGB-54 × IBWL but showed difference with parental hybrid. And the dhs of IGB-54 × PR, IGB-54 × KT and IGB-17 no variation exist in Fr- S, Fr-Curve; Fr- under C ie round in IGB-54 × PR, medium long (IGB-54 × KT, long fruit(IGB-17) shape, no curve(IGB-54 × PR), slight curved(IGB-54 × KT) and curved fruits in IGB-17, Fr-under C all parents had intensity of colour under calix was white. In genotype IGB-54 × IBWL, IGB-54 × PR, IGB-54 × KT, IGB-17 plants beard fruit set in 67.39%, 73.85 %, 44.83%, and 42.86% of the plants and remain without fruit set. But in case IGB-17 × KT, IGB-17 × PS, IGB-54 × PS, IGB-55 × PS, KS and BRLVAR-1 had no fruit set.
Among Invivo colchicine application in Si4 derived dhs had better response for quantitative traits in 10 crosses. Short plant height, delayed flowering, reduction in corolla length noticed in Si6 (0.1%c) in IGB-17 × KT due increased colchicine conc.0.02-0.1c.Some detrimental effects noticed at 0.1% c in ten genotypes vize., treated portion scorching (leaf scorching IGB-54 × KT, IGB-54 × PR), reduction in plant height (IGB-54 × KT, IGB-17 × KT), leaf size, flower and anther size(IGB-54 × PR, IGB-17 × KT), color of anthers(IGB-17×KT-greenish yellow) respectively.
Description
“Development of double haploids for production of bacterial
wilt resistance and high yielding brinjal (Solanum melongena
L.) lines by using anther culture Technique”