Sugarcane plays a significant role in sugar industry and is being grown on 0.96 million hectares with a production of 42 million tones in Pakistan. Unfortunately, its production is lagging behind due to biotic and abiotic stresses. Diseases like Red rot, Pokka boeng, Smut, Mosaic and Red stripe are some of the major biotic stresses which reduce the yield by 10-77%. Among these diseases, red rot disease of sugarcane caused by Colletotrichum falcatum is the most dreaded disease which has wiped out several important varieties from cultivation. The most satisfactory, dependable, long lasting and economical- means of lighting red rot is the use of resistant varieties. The conventional breeding program is much less effective and takes very long time for combining desirable traits. Because of non or sporadic flowering, natural viable fertile seed production has ever been a problem in Pakistan. Therefore, variability resulting from sexual crossing mayor may not provide sufficient room to permit greater chances of improvement in important characters of the crop.
Techniques such as induced mutation and ill vitro screening are being employed to create the new genetic variability to rectify specific defects and to improve specific desirable traits of highly adapted and genetically balanced sugarcane cultivars.
In present investigation, ill vitro techniques were successfully utilized for the production of red rot resistant plants of two varieties of sugarcane Le. CP 77,400 and BL-4.
The results of present investigation demonstrated that inner fresh leaves and shoot apical meristem of sugarcane were highly amenable to in vitro callus culture. Among the auxins, 2,4-D was more potent for callus induction and its subsequent growth. The optimum concentration of 2,4-D for callus induction was 3.0 mg/I. Low concentrations of 2,4-D were not effective and relatively higher concentrations retarded the callus initiation and growth. Effect of auxin-cytokinin interactions were not significant with respect to callus formation. All concentrations of 2,4-D with either BAP or Kinetin and NAA with BAP did not cause any promotive effect on callus induction and growth, rather an inhibiting effect was observed. For prolonged maintenance and curtailing the morphogenic capability, subsequent decrease in 2,4-D level was essential. Two major type of calluses developed from the cultured explants: dry nodular compact and smooth compact. One or both type of calluses could be obtained from any of the explants. Most abundantly formed callus was dry nodular and compact which was white to yellowish white in colour and was highly morphogenic. The optimum temperature for callus induction and proliferation was found to be 27 ±1°C. A significant increase in the rate of callus initiation and proliferation was observed in cultures incubated in dark as compared with light grown cultures.
In vitro regeneration in sugarcane callus was obtained via organogenesis and somatic embryogenesis. Organ differentiation capacity of various calluses was determined. Although regeneration was also obtained under hormone free conditions but best regeneration response was obtained in hormone mediated conditions. Regeneration response of both the varieties of sugarcane was different under different hormonal concentrations. In CP 77,400, best shoot induction response was obtained on MS medium supplemented with 1.0 mg/I BAP while in BL-4, MS medium containing 0.25 mg/I BAP + 0.25 mg/I Kinetin showed best organogenic response. The maximum number of shoot were induced in calluses of age 11-12 week while in sixteen weeks old calli, potential for shoot differentiation was dropped for all explants. Vigorous rooting of plantlets in both the varieties of sugarcane was induced after seven days of inoculation on MS medium supplemented with 2.0 mg/I NAA.
Somatic embryogenesis was obtained either directly or indirectly via intervening callus. After 14-18 days of incubation on MS medium containing 3.0 mg/l 2,4-0 + 0.25 mg/I BAP initiation of embryos from cut edges of the explant was observed. Initially transparent proembryoids appeared which gradually transformed into globular proembryoids. Some of these embryos developed into plantlets. Induction of direct embryo and frequency of embryogenesis was exclusively dependent on 2,4-0 and BAP concentrations. By an increase or decrease in the concentration of either 2,4-0 or BAP, the rate of embryogenesis was decreased. The highest incidence of embryogenesis was recorded in explants incubated at 3.0 mg/I 2,4-0 + 0.25 mg/I BAP. The embryogenic callus proliferated in to bipolar embryos. Maximum induction and proliferation of embryogenic callus was observed at 1.0 mg/I 2, 4-0 + 0.25 mg/I BAP in CP 77,400 and 1.0 mg/I 2, 4-0 + 0.5 mg/I BAP in BL-4. Germination response of embryos was dependant on cytokinin concentration and incubation period. In, CP 77,400 highest-frequency of regeneration was obtained on MS medium containing 1.0 mg/IBAP while in BL-4 it was on MS medium containing 1.0 mg/I BAP + 0.5 mg/l Kinetin.
Direct regeneration potential of apical meristem was studied on 12 different media designated AM1 to AM12 with different concentrations of BAP either alone or in combination with Kinetin or GA3. The response of cultured meristem in each medium varied in term of frequency and pattern of differentiation. In case of CP 77, 400, best shoot formation response was obtained in AM2 medium i.e. MS medium containing 1.5 mg/I BAP while in BL-4 100% shoot formation was obtained in AM6 medium(0.5 mg/I BAP+0.25 mg/I kinetin) within 10 days of inoculation with 1.7 shoots per explant. This medium promoted the formation of both solitary shoots and cluster of axillary shooots. Regeneration capability of cultured meristem was highly dependent on the size of cultured meristem. Meristem sizes from 0.5-5.0 mm were used in this study. Best apical shoot survival and regeneration ratio was harvested when meristem size was 4.0 and 5.0 mm. Rapid sugarcane clonal propagation was obtained successfully through apical meristem.
The major objective of the study was to induce mutation for red rot resistance. Therefore, for in vitro mutagenesis both chemical mutagen (Sodium azide) and gamma radiations were used. Well developed calli of seven week age and somatic embryos were treated with different doses of sodium azide ranging from 1.0-5.0 mg/I for five days. Proliferation response of callus was adversely affected at all concentrations of sodium azide. By increasing the concentration of sodium azide rate of callus proliferation and plant regeneration was decreased and at 5.0 mg/I concentration more than 50% callus became necrotic in CP 77,400 and 34% in BL-4.
In present study both calli and somatic embryos of CP 77, 400 were found to be more sensitive to sodium azide HS compared to BL-4.
When well developed eight week old calli and somatic embryos were treated with gamma rays it was noticed that exposure of 10 and 20 Gy enhanced the proliferation response of callus. At 30, 40 and 50 Gy exposures, callus proliferation and plant regeneration response was decreased and rate of necrosis was increased.
After treatment with sodium azide and gamma radiations, 10 week old differentiated and embryogenic calli were further inoculated on regeneration medium supplemented with different concentrations of partially purified toxin of Colletotrichum falcatum ranging from 0.05% to 0.50%. The plants surviving at high levels of toxin were selected as Ines resistant against red rot disease. Well developed plants were hardened in the green house. and were brought to field for field trials and study of red rot resistance according to disease rating scales. Total 164 regenerated plants of CP 77,400 and 196 regenerated plants of BL-4 after in vitro screening were brought to the field for R generation and were screened against two isolates of Colletotrichum falcatum. In R generation, 28 plants of CP 77, 400 and 22 plants of BL-4 showed red rot resistance response. Rest of all the plants showed varying degree of susceptibility. The plants showing resistance in R generation were again multiplied next year for R1 generation to check the stability of resistance. In R1 generation only 8 plants of CP 77, 400 and 5 plants of BL-4 showed stability in red rot resistance response against both the isolates of Colletotrichum falcatum.
The study was further supported by using various biochemical and molecular markers. Biochemical investigations of selected red rot resistant lines and control plant of both the varieties were undertaken. Quantitative analysis of soluble protein contents, total phenolic compounds. and some phenolic related enzymes (Phenyl alanine ammonia lyase, polyphenol oxidase) and both qantitative and qualitative analysis of peroxidase and esterases were carried out. Activities of soluble protein contents, total phenolic compounds and all the enzymes were found higher in the fresh leaves of selected resistant line of both the varieties of sugarcane. The electrophoretic pattern of peroxidases and esterases were also in conformity with activities of these enzymes which further confirm our results.