| Agriculture is the backbone of the national economies of many developing countries. Because of this production as well as productivity has to be increased to fulfill the national demand of crops particularly in developing countries. Pear is one of the most important temperate fruit crop that belongs to the Rosaceae family and Pomoideae sub-family and is one of the oldest fruit crops cultivated in temperate and sub-tropical regions of the world. China alone produces more than 60 % of the world's pears and the history of pear growing in China is over 4000 years.Among the several main factors, which are responsible for the reduction of pear production and productivity, is insect infestation. Crop losses due to insects are very high throughout the world. A huge amount of money is spent each year to control insects by chemical means and is environmentally unfriendly in many ways. To control insects in the pear orchard in environmentally friendly ways is not easy. One of the best methods to overcome the problem of insects is to incorporate insect resistance genes into the genome of the crops, which is not easy and feasible, by using traditional crop breeding methods. Moreover, traditional crop breeding methods are tedious and time consuming. However, it is possible to incorporate the insect resistance genes in a short period of time by using molecular methods.There are several types of insect resistance genes like Bt gene, CpTI gene, etc. In this experiment CpTI gene was used to transfer it into the genome of 6 cultivars of pear by using the biological vector Agrobacterium tumefasciens. The transformation of genes from one species to another species involves several steps like extraction of genes of interest from donor species, cloning it into the suitable vector, identification and multiplication, etc. In the same manner, a protocol has to be developed for the plant system regeneration after gene transfer which is very important, crucial and a basic step for success. Each species of plant and variety vary in its regeneration system. In the case of the pear several steps of tissue culturing has to be followed for the development of adventitious shoots from the explants with targeted genes. The following results were achieved from several experiments.1. 6-BA influenced greatly the shoot proliferation. In an average group-I, the cultivars i.e. Yali (P≤001), Aikansui (P≤001) and Whangkeumbae (P≤01) produced more than 7 times the new shoots during subculturing from one explant (7 to 8 new shoots per explant) in 1.5 mg L-1 6-BA and 0.1 mg l-1 DBA than group-II cultivars i.e. Abbe Fetel, Red Bartlet and Packham's Triumph from the same amount of media and hormone, which had produced only a maximum of 4 new shoots per explant which was not significantly different from other treatments. On the other hand, in all cultivars in both groups, only 3 to a maximum of 4.5 new shoots were found from one shoot from 0.5 mg L-1 6-BA and 0.1 mg L-1 IBA. From this experiment it is clear that the Asian pears cultivars (group I) are greatly influenced by the concentration of 6-BA than the European cultivars (group II).2. Regeneration of adventitious shoots from the leaves and other explants is essential, without which it is not possible to develop the transgenic crops. A complete protocol for adventitious shoot regeneration was developed from the leaves of four pear cultivars grown in vitro, Abbe Fetel, Yali, Packham's Triumph and Aikansui and one Chinese pear rootstock cultivar Tuli. A combination of TDZ and NAA had a significant effect on the number of shoot regenerations in all 5 tested cultivars. The maximum mean number of shoots and maximum number of shoots per leaf were obtained from the Yali cultivar: 11.8 (P<0.001) and 22, followed by the Aikansui and the Tuli 6.6 (P<0.001), 4.6 and 8 (P<0.001) and 12, respectively from 0.2 mg L-1 NAA combined with 1.0 mg L-1 TDZ. Similarly from Packham's Triumph and Abbe Fetel, the maximum mean number of shoots and maximum number of shoots per leaf were 5.6 (P≤0.001), 4.8 and 8 P≤0.001) and 11 respectively from the combination of NAA (1.0 mg L-1) and TDZ (2.0 mg L-1). Likewise, Abbe Fetel was the only one cultivar which had produced significantly higher adventitious shoots from the other two different combinations of 1.0 mg L-1 NAA and 5.0 mg L-1 6-BA (P≤0.05) and 2.0 mg L-1 NAA and 5.0 mg L-1 6-BA (P≤0.01) respectively. Some of the prominent problems associated with shoot proliferation and regeneration were also observed.3. The Whangkeumbae pear in the MS medium with several combinations of hormones was unable to produce adventitious shoots from its leaf explants; therefore an experiment was conducted to regenerate adventitious shoots from the stem explants. The total numbers of shoots were 87 (P≤0.001) per treatment, mean number of shoots per replication was 17.4 and a 100 % shoot regeneration rate was obtained from the stem explants with leaves. A maximum of 21 adventitious shoots were also obtained from one stem from the same treatment. Likewise, stem explants without leaves produced only a total number of 25 (P≤0.05) shoots with a mean number of 5 shoots per replication and with a 57 % shoot regeneration rate. A maximum of 5 adventitious shoots were also found from one stem explant from the same treatment.4. Mortality rates of explants during the initial phase of tissue culturing were found very high during the summer particularly from the shoot tip explants. A thorough investigation on the browning problem of the Yali Aikansui and Abbe Fetel pears was done and their control measures are suggested. According to the experiment results Yali was found more severely infected by browning than the other two cultivars. Similarly, it was also found that, the intensity of browning was less during spring and increased with time and reached its maximum during the summer. Shoot tips of Yali were found more severely infected by browning than the second node and other nodes and it accelerated the mortality rate up to 81 % of shoot explants during the summer months of July and August. Browning was greatly influenced by water soluble polyphenols, more of which were found from the shoot tip explants from the field (significantly higher at P≤0.05 and P≤0.01) than the other explants: second node and other nodes. Total polyphenol contents were found less in the explants from plantlets grown in vitro and less browning of medium appeared resulting in a lower mortality rate of explants. Collections of explants during early spring (P≤0.05 and P≤0.01) and use of other nodes rather shoot tip as explants (P≤0.05 and P≤0.01) were found to be better to prevent the browning problem. Finally, for the curative measures to control browning, use of ascorbic acid at the rate of 100 mg·L-1 medium (P≤0.05 and P≤0.01), 0.02 % polyvinyl pyrrolidone (PVP) in the cultural medium (P≤0.05), 96 hours dark treatment of other nodes (P≤0.05), and 12 hours cold treatment of explants at 4℃(P≤0.05 and P≤0.05) prior to sterilization of explants, were found to be the best way to control browning and therefore to increase the survival rate of cultured explants of the Yali pear.5. CpTI gene was found transformed into the genome of 2 pear cultivars Duli (4 plants) and Aigunsui (3 plants) after PCR amplification with primers and gel electrophoresis; however the transfer of the CpTI gene has to be confirm by further tests.6. Several types of problems were encountered during tissue culturing and gene transformation experiments. Some of the major problems were infection by bacteria, fungus, insects, etc. Vitrifiction and browning of shoots were also found severe in some cultivars. Some physiological problems like burr knots, abnormal petiole and extra callus were also found in some cases. The abnormal petiole and extra callus formation described by us is the first time it has been observed. Likewise, some cultivars were difficult to proliferate and regenerate its adventitious shoots. Similarly, it was also difficult to control the over growth of A. tumefasciens and the effect of CFE on adventitious shoot regeneration and newly grown shoots were detrimental. Finally the growth of A. tumefasciens was ceased when it was stored for longer period of times in the refrigerator.
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