Apple (Pyrus malus L.) cv. Red delicious belonging to Rosaceae family is a hard woody tree species that grows mainly in temperate and subtropical regions with minor production in higher altitudes of tropical regions (Chadha and Awasthi, 2005). Propagation of Apple is usually done by budding and grafting as the seeds are incompatible to produce true-to-type plantlets. Biotic stress caused by Venturia inaequalis in the form of scab disease is a major havoc in the cultivation of this crop as the disease not only lowers the yield but it spreads through air-borne spores and damages the upcoming crop through secondary infections. In the present study an attempt has been made to develop an efficient protocol for high frequency regeneration, proliferation, Agrobacterium mediated transformation of Apple cv. Red delicious to improve the scab resistance through ectopic expression of endochitinase gene from Trichoderma harzianum. Present study deals with the alternative propagation method for these valuable resources i.e. plant tissue culture. Plant tissue culture is a totipotency based multistep procedure and found suitable for successful propagation of many woody species globally. Problems related with the severity of scab disease in different Apple growing states requires an urgent need to combat the destructions caused by the pathogen and to generate protocols towards transgenic approaches to develop scab resistant varieties.
Before going for in vitro propagation study, different explants viz., leaf, cotyledonary node were collected from seedlings transplanted in the polyhouse of Department of Biotechnology, Bhimtal and given necessary treatments i.e. sterilization before inoculating them on MS media. In vitro propagation was done by two methods viz., through callus induction (indirect method) and through cotyledonary node (direct method) using explants from transplanted seedlings. In this experiment, effect of various PGRs in terms of percent callusing (callus induction frequency) was studied by inoculating leaf segments on MS medium supplemented with auxins and cytokinins in different concentrations in dark conditions for few days. Among the different concentrations of auxins and cytokinins used, maximum percent callus induction (91%) was observed on the media fortified with (NAA+2,4-D+BA+TDZ) (5.37+4.53+8.88+14.5) µM TDZ where callus induction was started within 11-12 days after inoculation of leaf explants. The callus obtained was dark green in colour. 25 days old callus was subjected to shoot induction medium supplemented with different PGRs. Findings of this experiment showed that BA+NAA+GA3+2-iP (5.0+1.0+3.0+39.35) µM as the best combination for shoot induction and proliferation from leaf derived callus after 25 days of incubation as the number of microshoots generated were not only quite high (10.667±0.333 microshoots/callus) but they were also having average shoot length upto (8.700±0.173) cm with average number of leaves per shoot (68.333±3.283). To minimize the chances of somaclonal variations, in vitro study was also done by cotyledonary node as explants. Effect of two different combinations of PGRs was studied viz., BAP+GA3+IBA and BAP+NAA+GA3+2-iP which showed almost similar shoot induction rate. The best combination of PGRs among these two was BA+GA3+IBA (4.44+2.89+0.5) µM in which shoot induction occurred within three days with (2.667±0.333) shoots per explant and the number increased to sixteen in just fifteen days with (96.294±3.706) % shoot induction rate. Similarly, BAP+NAA+GA3+2-iP (4.44+0.537+1.445+39.36) µM was able to induce fifteen shoots per explant in fifteen days starting from (1.667±0.333) shoots within three days with (94.997±0.96) % shoot induction rate. Shoot elongation and proliferation was done on the same combination of PGRs where MS medium supplemented with BA (4.4 µM), GA3 (2.89 µM) and IBA (0.5 µM) was considered as the best shoot proliferation medium, when observed after fifteen days of shoot induction as it contributes higher mean number of shoots (18.000±1.155), higher average number of leaves (122.333±5.239) and higher shoot forming capacity (SFC) index (15.9) per explant.
However, the average length of shoots per explant (9.333±0.240 cm) was higher in BA+NAA+GA3+2-iP (4.44+0.537+1.445+39.36) µM as compared to BA+GA3+IBA (4.44+2.89+0.5) µM where it was (9.133±0.133) cm. For root induction, in vitro raised microshoots were subjected to half strength MS medium supplemented with IBA+IAA (4.90+2.855) µM which was found to be most effective and induced maximum number of average roots per explant after 25 days of inoculation in ½ MS medium. After complete plantlet was developed in vitro it was transferred to thermacol cups containing a mixture of sterile soil, sand and farmyard manure mixture (3:1:1) and acclimatized in culture room then in green house and finally hardened in polyhouse under ex situ conditions. In this way in vitro raised and subsequently hardened plantlets were developed which were further used as explants for transformation studies.
For standardizing the transformation protocol, leaves, callus and internodal stem were used as explants and influence of different parameters viz., effect of pre-culture prior to infection, Agrobacterium concentration, infection and co-cultivation time, kanamycin, cefotaxime, acetosyringone, respectively, on transformation efficiency of the three explants was investigated individually. It was observed during the study that pre-culture of explants was not necessary as more number of explants died while pre-culturing than explants that were infected immediately after being excised. Agrobacterium concentration of O.D.600 – 0.8 was found appropriate for the working bacterial suspension culture as the transformation efficiency was highest at this concentration of all the explants tested. In the present study seven different infection time periods were applied to explants (leaf, callus and internodal stem) co-cultivated for three different time regimes (24, 48 and 72 hrs), respectively, for the production of transgenic plants. 72 hrs co-cultivation was best duration reported for each of the three explants used because the % transformation efficiency was comparatively higher than that obtained during 24 and 48 hrs. Infection time was found a bit longer for intermodal stem explants (6 hrs) as compared to leaf and callus explants (1 hr). Kanamycin at 100 mg/l showed complete mortality of explants (explants survival rate was 0 %) while there was 90 % regeneration in absence of kanamycin. Therefore, selection at this concentration was maintained upto five successive shoot subcultures and also applied to root induction and proliferation medium.
500 mg/l was found to be the best concentration of cefotaxime at which the excessive growth of Agrobacteria was eliminated out and maximum regeneration frequency (26 %) was reported. The results of the present study revealed that there was not any significant difference between the experiments where acetosyringone was used as an inducer or not. Percent transformation efficiency obtained in stem while using acetosyringone during infection time was much nearer (26.663 %) to the control experiment (26.050 %). Similarly, concentration of acetosyringone used during the co-cultivation period resulted in 26.273 % transformation efficiency. Although, the values are slightly higher but are insignificant with respect to control values. Moreover, when acetosyringone was provided both during the infection as well as co-cultivation phase, there was a noticeable reduction in the value of % transformation efficiency (22.947). So, it was concluded that presence or absence of acetosyringone during infection or co-cultivation period was not essential. Employment of these parameters helped in successful integration of T-DNA in Apple plants which were further verified by PCR amplification of genomic DNA using endochitinase and nptII gene specific primers. An endochitinase fragment of 510-517 bp and nptII fragment of 190 and 153 bp was amplified through agarose gel. Nucleotide samples were sent for sequencing which further confirmed our study as the sequences showed 91- 99 % identity on BLAST with endochitinase gene sequence in the NCBI database.
In the present study, in vitro evaluation of increased fungal resistance in Apple plants against the scab pathogen Venturia inaequalis was done using the crude protein extracts of control and transformed plants as well as by detached leaf assay. Enzymatic activity vary according to the total protein present in the leaf tissue. Specific activity varied from 1.651 to 2.499 nmol/mg/min. Transgenic plants showed 3.26 to 4.93 fold more chitinolytic activity as compared to control. In vitro pathogen inhibition assay revealed that inhibition ranged between 43.91- 47.39 % at 100 µg concentration, 58.04- 61.49 % at 250 µg concentration and at 500 µg concentration it ranged from 69.90 to 73.55 % when compared with controlled plant extract. Similarly, inhibition ranged between 56.62- 59.56 % at 100 µg concentration, 68.52- 73.24 % at 250 µg concentration and at 500 µg concentration it ranged from 81.54- 85.60 % when compared with blank (sterile water) as extract. Inhibition per cent rate of transgenic plant 2 was observed to be more. Further, it was observed that, among all PCR positive plants only transgenic plant 2 showed higher chitinolytic activity. In the detached leaf assay, transgenic plants showed much less symptom of V.inaequalis and symptoms were restricted to inoculated region whereas in non transgenics, spreading of necrosis and chlorosis of almost whole leaf was observed. Also decaying of the whole leaf was observed whereas leaf from transgenic plant was green and healthy. Leaf area infected in control was almost greater than 50 %, while in transgenic plants it ranged from 1 to 30 %. The efficacy of the gene construct was demonstrated further through inhibition of pathogen (V.inaequalis) growth in the detached Apple leaves of transformed and untransformed plants. The transgenic Apple leaves did not develop infection even after 10 days of inoculation with V.inaequalis as against 5-6 days for disease development in untransformed leaves. Transgenic plant 1, 2 and 3 showed disease-rating scores of 3.1, 2.5, and 3.5 as an average score of three experiments, respectively, versus a score of 4.9 for the non-transformants. These results indicate that the three transformants exhibited partial resistance to V.inaequalis, which was sufficient to delay the spread of lesion areas of the disease. The degree of disease symptoms correlated well with the level of chitinase enzyme i.e., the transgenic plants with a higher level of chitinase activity tended to have a smaller total lesion area. All the transformants took longer period for the development of necrosis among which Transgenic plant 2 took the maximum time i.e. 30 days. Thus, the present study concludes that endochitinase gene integrated into the host plants showed its effect by minimising the severity of the scab disease. Scab disease in Apple caused by the fungal pathogen Venturia inaequalis is one of the most serious diseases occurring worldwide. It has also been a major constraint for agriculture in hilly areas like Uttarakhand. Various approaches like genomic, proteomic, bioinformatic and modern genetic studies had been applied to study this woody plant-pathogen interaction (Bastiaanse et al., 2016). Our study concluded that transformation efficiency was comparatively higher in internodal stem (than leaf and callus) which were produced by direct regeneration through cotyledonary nodes. This strategy can be used further for obtaining true-to-type plantlets. Introduction of endochitinase gene in P.malus cv. Red delicious improved the fungal resistance in transformed Apple plants due to increased chitinolytic activity of the foresaid protein. Transformed Apple plants acquiring such gene showed very less symptoms and that too after a month or so. Leaves remained healthier and greener than the untransformed plants. Use of this transformation protocol could be applied on a large scale for the production of P.malus cv. Red delicious and distributing them to farmers of Uttarakhand for generating employment which will bring perceptible changes in the rural economy of the state. The protocol will serve as a boon in increasing productivity of this very important cash crop besides meeting the nutritional requirements of the entire population of Uttarakhand and also provides an easy alternative to farmers who were unable to cope earlier with the adverse situations due to lack of knowledge, money and either started migrating from hilly areas to plains or commiting suicides due to heavy losses of the food crops.
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