Introduction

Okra [Abelmoschus esculentus (L.) Moench] also called lady’s finger or bindi is a polyploid vegetable with chromosome number 2n=130 belonging to family Malvaceae. A number of insect pests and viruses affect the production of this crop. Whitefly (Bemisia tabaci) transmitted virus complex consisting of a monopartite begomovirus, bhendi yellow vein mosaic virus (BYVMV) causing Yellow Vein Mosaic Virus (YVMV) is the most important and destructive viral disease in okra (Rana et al., 2006). The total loss due to YVMV has been reported to be 20-30%, which could rise to 80-90% if the carrier of the virus is not controlled (Richardson, 1997). The use of chemicals and culling off the infected plants is not practical and economical solution to control the virus, and therefore, development of resistant/tolerant varieties seems to be the best option to curtail the loss. Undomesticated related species are the genetic stocks of resistant genes for different pest, diseases and abiotic stresses (Rattan et. al., 2015). Lack of stable source of resistance to YVMV in cultivated species is the major constrain in developing stable resistant variety in okra.  However, some of the wild species of okra have been reported to be stable and reliable sources of resistance to YVMV. One such wild species of okra, A. tetraphyllus is an important resistance sources to YVMV (Prabu, 2005), but the sterility problems and trouble in producing subsequent generations or even to carry out back crosses hampers the transfer of resistance from wild species. Hence  for the development of YVMV resistant lines adoption of non-conventional methods of breeding like plant tissue culture mediated introgression is an urgent need. Embryo rescue technique can be efficiently used to overcome post-zygotic incompatibility which is found to operate between these species. In view of this, the investigation was undertaken with the objective to transfer resistance genes from wild species (A. tetraphyllus IC 141017) to popular okra varieties mediated through embryo rescue.

Materials and Methods

Planting Material and Interspecific Hybridization

The planting materials used in the investigation comprised of four cultivated genotypes of okra [A. esculentus L. (Moench)], viz., Arka Anamika, Parbhani Kranti, Pusa Makhmali (procured from ICAR-NBPGR, New Delhi), Jammu okra-05 (procured from SKUAST, Jammu), and a wild species, A. tetraphyllus (IC 141017) procured from ICAR-Indian Institute of Vegetable Research, Varanasi. All these genotypes were sown in the Agriculture Farm of DAV University, Jalandhar in April, 2017. Reciprocal crosses were made between the cultivated and wild genotypes.  Female flowers were selected at the balloon stage a day prior to pollination and their bagging was done to avoid undesirable crossing. Next day they were pollinated with the desirable pollen.

Embryo Rescue and Shoot and Root Regeneration

Fruits of okra at immature stage were harvested at 5, 10, 15 and 20 days after pollination (DAP). The immature seeds were extracted from these fruits and they were inoculated on media composed of Murashige and Skoog (MS) medium containing different concentrations of 6-Benzyl amino purine (BAP) for embryo emergence or callogenesis. The emerged embryos (4-5 cm) were further cultured on different treatment combinations so as to study their effect on shoot emergence. These treatment combinations were MS + 0.25 mg l^-1 NAA + 0.5 mg l^-1 IBA; MS + 0.5 mg l^-1 NAA + 0.5 mg l^-1 IBA; MS + 1.0 mg l^-1 NAA + 0.5 mg l^-1 IBA; MS + 0.25 mg l^-1  NAA + 1.0 mg l^-1 IBA; MS + 0.5 mg l^-1 NAA + 1.0 mg l^-1 IBA; MS + 1.0 mg l^-1 NAA + 1.0 mg l^-1 IBA. Incubation of these cultures was done at 26±2⁰C for 30 d after which they were sub-cultured on to freshly prepared medium for shoot multiplication. These shoots were transferred then transferred to root regeneration medium having composition of half-strength MS medium +0.25 mg l^-1 IBA + 200 mg l^-1 and activated charcoal, for root initiation.

Hardening of the Plantlets

Well rooted plantlets were isolated from the test tube with utmost care so that the shoots do not get damaged. After washing them with distilled water to remove the adhering agar, the plantlets were transplanted in sterilized soil less media (cocopeat: vermiculite: perlite in ratio of 1:1:1) in the small pots. Watering of these plantlets was done at every 15 days with half-strength MS medium.

Results and Discussion

Interspecific Hybridization

The four cultivated varieties started flowering in the month of May 2017, whereas, the wild species (IC 141017) started flowering in the month of Sept. 2017. The main problem in the hybridization programme of the present study was non-synchronised flowering among cultivated and wild genotypes. The wild genotype started flowering by the Sept. 2017, however the cultivated genotypes which were sown in the April, 2017 stopped flowering by August, 2017.  So, when the cultivated genotypes were in their peak period of flowering there was no flowering in the wild genotype. To overcome this problem, repeated sowings of cultivated varieties of okra was done under controlled conditions so as to get a synchronous flowering.

Figure 1: Different interspecific cross combinations and wild genotype Click here to view figure

Table 1: Cross compatibility between Abelmoschus esculentus L. (Moench) and A. tetraphyllus IC 141017   

A total of 50 crosses were made in each cross combination, of the four cultivated genotypes, fruit setting was observed only in Arka Anamika, Parbhani Kranti and Pusa Makhmali when cultivated genotypes were used as female parents. Fruit setting failed when A. tetraphyllus (IC 14101) was used as female parent (Table 1). Similar results were observed by Mamidwar et al. (1979), Meshram and Dhapake (1981), Sheela (1986). They observed that fruit set was maximum when A. esculantus was used as female parent in an interspecific cross between A. esculentus × A. tetraphyllus. Small fruits were obtained in the all the cross combinations. The fruits appeared normal till 25 d after pollination, but after that the fruits showed splitting/cracking from the distal end. The seeds looked healthy for up to 10-15 d after pollination, after that it shrivelled and became pale yellow (Fig. 2). The findings are in time with Mamidwar et al. (1979) who also obtained seed less fruits and shrivelled seeds when crossed Abelmoschus esculentus × Abelmoschus tetraphyllus.

Figure 2: (a to d):a) Small interspecific hybrid fruit b) Distal cracking in the fruits after 25 DAP c)Small white yet succulent seeds after 15 DAP d)Degeneration of seeds after 25 DAP Click here to view figure

Sureshbabu and Datta (1990) also observed the similar results and inferred that slow pollen tube growth, abnormal pollen tube, and abortion of fertilized ovules or scarcity of pollen grains could be the reason for no seed formation in interspecific hybrids. The findings are also in time with Sindhu (1993) who obtained shrivelled seeds in interspecific hybridization which may be attributed to the poor development of endosperm. Age at which embryo is extracted for inoculation affects a lot in regeneration of plant from embryo. The excision stage of embryo varies with crop. After 5, 10, 15 and 20 d of pollination, the fruits were harvested in the present study. This could be due to the fact that the embryo of the seeds which were harvested before 15 d of pollination were quite immature for pollination however, the embryo of the seeds which were harvested after 15 d of pollination were degenerated. Similar findings were reported by Rajamany et al. (2006), who obtained interspecific hybrids o A. esculentus × A. moschatus when excised embryos at 12 and 15 d after pollination through embryo rescue technique.

Effect of Media of Embryo Emergence

The fruits of the all the cross combinations which were harvested at 5, 10 and 15 d, had seeds which were shiny and succulent yet small and white. As the number of days after pollination increased the seeds started appearing shrivelled and dull white (Fig. 2). However, the seeds remained succulent at 15 d after pollination. Inoculations of these harvested seeds were done on different treatment combinations viz., MS + 0.25 mg l^-1  BAP; MS + 0.5 mg l^-1  BAP; MS + 1.0 mg l^-1  BAP; MS + 1.5 mg l^-1  BAP. The results pertaining to this component is presented in Table 2. Cross combinations, Arka Anamika × A. tetraphyllus IC 141017 showed emergence of embryo 25 d after inoculation when inoculated media containing MS + 0.25 mg l^-1  BAP. Cross combination, viz., Pusa Makhmali × A. tetraphyllus IC 141017 and Parbhani Kranti × A. tetraphyllus IC 141017 showed direct emergence of embryo 30-35 d after inoculation in the same media. Emergence of embryo was observed in all the cross combinations after 45-50 d of inoculation in the MS media containing BAP @0.5 mg l^-1. No cross combinations showed embryo emergence in media comprising of MS + 1.0 mg l^-1  BAP; MS + 1.5 mg l^-1  BAP.

Table 2: Response of different interspecific hybrids on growth media

Role of media is major in embryo rescue, as it acts as endosperm by providing nutrient to the excised embryo. The seeds of fruits harvested after 15 d of pollination were inoculated in MS media containing various concentrations of BAP. MS media containing 0.25 mg of BAP gave the best results in all cross combinations. These findings corroborates with the results of Kabir et al. (2008), who observed good regeneration with BAP. It was observed that lower the concentration of BAP greater were the results with early emergence of embryo. Negative effect on plant regeneration with higher concentration of BAP was also observed by earlier researchers viz.,  Kashif Waseem et al. (2011) and Zayova et al. (2012).

Regeneration and Hardening Plantlets

Shoot Regeneration

Different treatments such as MS + 0.25 mg l^-1  NAA + 0.5 mg l^-1  IBA; MS + 0.5 mg l^-1  NAA + 0.5 mg l^-1  IBA; MS + 0.5 mg l^-1  NAA + 0.5 mg l^-1  IBA; MS + 0.25 mg l^-1  NAA + 1.0 mg l^-1  IBA; MS + 0.5 mg l^-1  NAA + 1.0 mg l^-1IBA; MS + 1.0 mg l^-1  NAA + 1.0 mg l^-1  IBA were used to subculture the two true leaf stage emerged embryos. MS media supplemented with 0.5 mg l^-1  NAA + 1.0 mg l^-1  IBA  resulted in  the largest number of shoots per explant (0.92±0.12%) with the maximum frequency of shoot regeneration (86±0.12%) in Parbhani Kranti × A. tetraphyllus IC 141017.  The same media combination revealed maximum number of shoots/explants to the tune of 0.89±0.10 and 0.88±0.13 in cross combinations viz., Arka Anamika × A. tetraphyllus IC 141017 and Pusa Makhmali × A. tetraphyllus IC 141017 respectively. Similarly, the maximum frequency of shoot regeneration was observed in Arka Anamika × A. tetraphyllus IC 141017 (85±0.11%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.14%) on this media combination (Table 3).  Kabir et al. (2008) also obtained shoot differentiation in okra when MS media was supplemented with NAA and Dhande et al. (2012) who observed that MS media supplemented with IBA and NAA gave good shoot regeneration in okra.

Table 3: Shoot regeneration in different media combinations

Root Regeneration

The subculturing of regenerated shoots was done in the media containing different levels of auxins. After 10-15 days of subculturing there was initiation of roots in all cross combination and well developed roots were seen in 3 weeks. Arka Anamika × A. tetraphyllus IC 141017, Pusa Makhmali × A. tetraphyllus IC 141017, Parbhani Kranti × A. Tetraphyllus IC 141017 showed the maximum root regeneration  to the tune of 56.08, 78.28, and 48.12%, respectively in half-strength MS media containing 0.25 mg l^-1  IBA + 200mg l^-1 and  activated charcoal. Plantlets after proper rooting were transferred to sterilized soil less mixture (cocopeat: vermiculite: perlite in the ration of 1:1:1) in pots as presented in Fig. 3.

Figure 3: (a to h): a) Immature seeds, b)Inoculation of immature seeds, c)Emergence of embryo from immature seed, d)Development of callus from embryo, e)Development of embryonic callus, f)Shoot and Root development g)Acclimatization of invitro development plantlets. Click here to view figure

Introduction of roots on regenerated shoots is important for establishment of plantlets in soil Kabir et al (2008). Maximum roots were observed in MS media containing 0.25mg l^-1  IBA + 200mg l^-1  and activated charcoal in the present study. Kabir et al. (2008) also reported good root regeneration by supplementing the MS media with IBA. Activated charcoal was used as anti browning agent. Activated charcoal stimulates nitrogen uptake by shoots and induce a dark environment resulting invitro rooting (Thomas, 2008). However, Muhammad Isshad et al. 2017 suggested that the reduced salt concentration to be effective for invitro rooting due to reduced nitrogen content rather than reduced osmotic potential.  The results also corroborates with Mohammad Irshad et al. (2017) who used 1/2 MS supplemented with IBA and AC (Activated Charcoal) for rooting in okra.

Conclusion

Interspecific hybridization using wild relatives through biotechnological interventions can be the best way to introduce desirable genes absent in the domesticated species. Present study was made with an aim to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Out of the four popular cultivars used in crossing, fruit set was observed only in Arka Anamika, Pusa Makhmali, Parbhani Kranti  when A. tetraphyallus IC141017 was used as a male parent. Healthy seeds can only be obtained before 15 d after pollination. Ms media containing on 0.5 mg l^-1  NAA + 1.0 mg l^-1  IBA  resulted in highest shoot regeneration in cross combinations, viz., Parbhani Kranti × A. tetraphyllus  IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017. Among these Parbhani Kranti × A. tetraphyllus IC141017 showed greatest number of shoots per explant (0.92±0.12) as well as the maximum frequency of shoot regeneration (86±0.12%). On MS media supplemented with 0.5 mg l^-1  NAA + 1.0 mg l^-1  IBA, the maximum frequency of shoot regeneration was recorded in Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%)

Acknowledgment

The authors are thankful to ICAR-National Bureau of Plant Genetic Resources (NBPGR), Indian Institute of Vegetable Research (IIVR), Varanasi and Sher-e- Kashmir University of Agricultural Sciences and Technology (SKUAST),-Jammu for providing the germplasm for conducting the present investigation. The authors are also highly thankful to Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India for funding the research project entitled, “Alien introgression of Yellow Vein Mossaic Virus (YVMV) resistance genes in okra (Abelmoschus esculentus L) through embryo rescue technique.” under Extra Mural Scheme.”

Conflict of Interest

There is no conflict of interest among authors for honorarium, grants, membership, employment, ownership of stock or any other interest or non‐financial interest such as personal or professional relation, affiliation and knowledge of the research topic of the present research paper.

Funding Source

The present investigation was carried out under the Extra Mural Research project funded by the Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India.

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