Development of Interspecific Hybrids (Abelmoschus esculentus × A. tetraphyllus) in Okra using Embryo Rescue Approach

Undomesticated related species are the rich stock of genes providing resistance to various diseases, pests and unfavourable environmental conditions. The sexual incompatibilities are bottleneck in introgression of such genes from wild species to popular as varieties. The present study was executed to standardize a protocol of embryo rescue so to obtain the hybrids of Abelmoschus esculentus × A. tetraphyllus IC141017. Crosses were made between four (Arka Anamika, Pusa Makhmali, Parbhani Kranti, Jammu Okra-05) cultivated varieties of okra (A. esculentus) and a wild species (A. tetraphyllus IC141017) in reciprocal manner. Out of the four popular cultivars used in crossing, fruit set was recorded in Arka Anamika, Pusa Makhmali, Parbhani Kranti only when A. tetraphyallus IC141017 was used as a male parent. Fruit showed distal cracking after 25 d of pollination. The seeds appeared healthy for up to 15 d after pollination and later shrivelled and became pale yellow. Cross combinations, viz., Parbhani Kranti × A. tetraphyllus IC141017, Arka Anamika × A. tetraphyllus IC141017 and Pusa Makhmali × A. tetraphyllus IC 141017 showed the highest shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. Maximum number of shoots per explant (0.92±0.12) as well as the maximum frequency of shoot regeneration (86±0.12%) was reported in Parbhani Kranti × A. tetraphyllus IC141017. Arka Anamika × A. tetraphyllus IC 141017 (86±0.12%) and Pusa Makhmali × A. tetraphyllus IC 141017 (82±0.13%) resulted in high frequency of shoot regeneration on MS media containing 0.5 mg l-1 NAA + 1.0 mg l-1 IBA. It can be inferred that MS media containing on 0.5 mg l-1 NAA + 1.0 mg l-1 IBA showed positive results in regeneration of interspecific hybrids of okra.


Okra [Abelmoschus esculentus (L.)
Moench] also called lady's finger or Bhindi 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.

P l a n t i n g M a t e r i a l a n d i n t e r s p e c i f i c hybridization
The planting material 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 during 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±2 0 C for 30 d after which they were sub-cultured on to freshly prepared medium for shoot multiplication. These shoots were 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 small pots. Watering of these plantlets was done at 15 days interval with half-strength MS medium.

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 September 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. 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 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 line with Mamidwar et al. (1979) who also obtained seed less fruits and shrivelled seeds when crossed Abelmoschus esculentus × Abelmoschus tetraphyllus.
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 line 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.

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 Fig. 3.
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 earlier work of 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 (IC 141017). Out of the four popular cultivars used in crossing, fruit set was observed only in Arka Anamika, Pusa Makhmali, Parbhani Kranti when A. tetraphyallus (IC 141017) was used as a male parent. Healthy seeds can only be obtained before 15 d after pollination. MS media containing 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 (IC 141017), Arka Anamika × A. tetraphyllus (IC 141017) and Pusa Makhmali × A. tetraphyllus (IC 141017). Among these Parbhani Kranti × A. tetraphyllus (IC 141017) showed maximum 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