NSave Nature to Survive

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1 NSave Nature to Survive 12(1): , 2017 (Supplement on Agronomy) EMBRYO RESCUE OF LOW CHILL PEACH HYBRIDS AS AFFECTED BY EMBRYO AGE AND GROWTH REGULATORS INDIRA DEVI*, HARMINDER SINGH AND ANIRUDH THAKUR Department of Fruit Science, Punjab Agricultural University, Ludhiana , Punjab, INDIA indu90-coafs@pau.edu KEYWORDS Growth stages WP media Growth regulators Peach hybrids Received on : Accepted on : *Corresponding author ABSTRACT Embryo rescue is an important technique to recover the hybrid seeds from the low chill peach hybrids. Low chill peach cultivars show low germination percentage because of immaturity of zygote. In present investigation fruits of different crosses (Shan-i-Punjab x Florda Prince, Shan-i-Punjab x Flordaglo and Shan-i-Punjab x Prabhat) were harvested at 65, 75 and 85 days of pollination and embryos were cultured in Woody Plant medium supplemented with various combinations of plant growth regulators. The effect of embryo age and growth regulators on embryo germination, shoot length and root length indicated that embryos harvested at fully matured stage (85 days after pollination) and cultured in M 2 medium [WP + BAP (0.25)+ IBA (0.05)] showed maximum embryo germination (81.01 %). Maximum shoot length and root length was also observed in 85 days aged embryo in M 4 [WP + BAP (1.00) + IBA (0.25)] medium (6.30cm and 7.88cm respectively). Among different crosses seeds of Shan-i-Punjab x Prabhat showed higher embryo germination (77.69 %) and maximum shoot and root length (5.79 cm and 6.47 cm respectively). The study concluded that embryo rescued at 85 days after pollination gave maximum germination and better growth of germinated embryo. INTRODUCTION Peach (Prunus persica L.) belongs to family Rosaceae and is well adapted to temperate and sub-tropical regions. It is native to China where its culture dates back at least 4000 years. In India, it is grown in the mid hills zone of Himalayas extending from Jammu and Kashmir to North Eastern States at an altitude of 1000 to 2000 m above MSL. Due to wider climatic adaptability it is now successfully cultivated in various subtropical regions of the world with the breeding efforts for low chill peach cultivars (Kumar et al., 2015). With the advancement of breeding efforts, low chill peach cultivars have been developed and these early ripening peach varieties gets advantages of marketing window and high profit. The early ripening trait is genetically controlled and is an important breeding objective in many stone fruit breeding programs (Anderson and Byrne, 2001). Poor germination of seeds of early - maturing peach varieties is a major obstacle in the development of early ripening peaches because of immaturity of the zygotic embryo in hybrid seeds (Mancuso et al., 2002). Thus, embryo culture technique is necessary to recover hybrid seeds. Embryo culture is useful for saving hybrids when there is poor embryo development or abortion. In early ripening peaches embryo abortion occurs because the flesh matures before seed maturity and thus seeds of early maturing fruits have low germination percentage. Embryo culture allows the germination of embryos of the seeds that fail to germinate in conventional planting conditions and can also shorten the breeding cycle by overcoming dormancy in seeds. In general, peach fruit require more than 105 days from full bloom to harvesting for embryo maturity (Bacon and Byrne, 2005) but early ripening varieties that have been bred in temperate fruit improvement programs have fruit development period of less than 90 days. It is an important breeding program where extreme early ripening is a major goal. Success of embryo rescue depends upon many factors such as genotype, developmental stage of embryo, basic composition of culture media, growth regulators and culture conditions. Centallas et al., (2000) studied the germination and development of early ripening peach seeds and observed that full embryo development was not reached at the time of fruit flesh ripening and when cultured in SBH medium showed high germination rate and good development. Uma et al., (2011) cultured zygotic embryos of banana at different maturity stages to determine the best maturity stage for embryo rescue and found that fully matured embryos regenerated directly into plantlets without producing callus whereas, immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration and 88 per cent survival in hybrid banana was achieved. In mango hybrids more than 80 per cent germination was obtained through embryo culture (Perez Hernandez and Grajal-Martin, 2011). Embryos of some fruits require a cold treatment of 4 C to break dormancy. The growth of sweet cherry embryos was possible only when immature and mature embryos were treated at 4 C cold treatment for 40 days and 60 days (Fathi et al., 2009). The optimum temperature for growth of embryos depends on plant species. Plant growth regulators play an important role in embryo culture and enhance the embryo growth (Jeengool and Boonprakob, 2004). The present investigation was conducted with the objective to determine the best development stage 499

2 INDIRA DEVI et al., for embryo rescue and to study the effect of growth regulator combinations on embryo culture of low chill peach hybrids. MATERIALS AND METHODS The present study was conducted at Fruit Research Farm and Tissue Culture Laboratory, Department of Fruit Science, Punjab Agricultural University, Ludhiana. The crosses were made between low chill peach cultivars viz. Shan-i-Punjab (( &) &) x Florda Prince (B&), Shan-i-Punjab (@&) x Flordaglo (B&) and Shan-i-Punjab (@&) x Prabhat (B&). Pollination of emasculated flower buds was done on the same day with stored pollen grains because flowering of Shan-i-Punjab cultivar do not coincide with Florda Prince, Flordaglo and Prabhat. The fruits from these crosses were harvested after 65 days, 75 days and 85 days of pollination. Seeds were extracted from the fruits under laboratory condition. Extracted seeds were surface sterilized with 70% ethanol for 2 minutes. Seeds were than rinsed in sterile water and treated with HgCl 2 (0.1 %) for different duration of time and than again washed 2-3 times with sterile distilled water. Seeds were dissected with transversal cut to separate the seed coat. Embryo with half of their cotyledons were excised under laminar air flow and cultured in test tubes containing culture medium (n=30). Culture media used for this experiment was Woody Plant medium supplemented with different concentration of BAP (0-2mg/L) and IBA (0-1mg/L). Seven medium were prepared i.e. M 0 (WP medium), M 1 (WP medium+ BAP 0.125mg/L + IBA 0.01mg/L), M 2 (WP medium + BAP 0.25mg/L + IBA 0.05mg/L), M 3 (WP medium+ BAP 0.50mg/L + IBA 0.1mg/L), M 4 (WP medium + BAP 1mg/L + IBA 0.25mg/L), M 5 (WP medium + BAP 1.5mg/L + IBA 0.50mg/L) and M 6 (WP medium+ BAP 2mg/L + IBA 1mg/L). After inoculating the embryo in culture medium the cultured test tubes were kept at 4 C for stratification. An embryo was considered as germinated after its radicle emerge out and grow into the medium e 5mm. After stratification the embryo cultured tubes were transferred to a growth chamber at 24 ± 2 C for germination. The data regarding the embryo germination percentage, shoot length and root length was recorded after 2 to 3 weeks of embryo culture in light. The experiment was laid out as factorial completely randomized design (CRD) with three stages of embryo growth and seven growing medium. Data were analyzed using SAS v9.0.0 software and means were compared using Least Significant Difference (Fisher s LSD) test at d 0.05 level of significance. RESULTS AND DISCUSSION The effect of embryo age and WP medium supplemented with growth regulators on embryo germination percentage of low chill peach hybrids indicate that embryo harvested from advanced maturity observed higher germination (Table 1). Maximum mean percentage of embryo germination (78.10 %) was recorded in M 2 medium [WP + BAP (0.25mg/L) + IBA (0.05 mg/l)] which was statistically at par with the embryo germination in M 1 medium [WP + BAP (0.125mg/L) + IBA (0.01 mg/l)] i.e per cent. The lowest germination percentage (71.00 %) was found in M 6 medium [WP+ BAP Table 1: Response of embryo age and WP medium supplemented with growth regulators on embryo germination (%) of low chill peach hybrids Treatments 65 DAP 75 DAP 85 DAP WP + Growth Regulators (mg/l) Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Mean x x x x x x x x x Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat M 0 : Control bc* 1 M : BAP (0.125) + IBA (0.01) ab 2 M : BAP (0.25)+ IBA (0.05) a 3 M : BAP (0.50)+ IBA (0.10) bc 4 M : BAP (1.00)+ IBA (0.25) bc 5 M : BAP (1.50)+ IBA (0.50) cd 6 M : BAP (2.00)+ IBA (1.00) d Mean Days mean c b a Crosses Mean Shan-i-Punjab x Florda b LSD Prince : Trt - Crosses 1.63 Days 1.63 Trt x crosses Trt x days Shan-i-Punjab x Flordaglo c Crosses x days Shan-i-Punjab x Prabhat a Trt x crosses x days * Values with the same letters are not significantly different according to Fisher s LSD test at 5% level 500

3 EMBRYO RESCUE OF LOW CHILL PEACH HYBRIDS AS AFFECTED Table 2: Response of embryo age and WP medium supplemented with growth regulators on shoot length (cm) of low chill peach hybrids Treatments 65 DAP 75 DAP 85 DAP MS + Growth Regulators Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Mean (mg/l) x x x x x x x x x Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat M 0 : Control bc* 1 M : BAP (0.125) + IBA (0.01) ab 2 M : BAP (0.25)+ IBA (0.05) ab 3 M : BAP (0.50)+ IBA (0.10) a 4 M : BAP (1.00)+ IBA (0.25) a 5 M : BAP (1.50)+ IBA (0.50) cd 6 M : BAP (2.00)+ IBA (1.00) d Mean Days mean 5.08 c 5.55 b 5.94 a Crosses Mean Shan-i-Punjab Florda 5.70 a LSD 0.36 x Prince 0.05 : Trt - Crosses 0.23 Days 0.23 Trt x crosses Trt x days Shan-i-Punjab x Flordaglo 5.28 b Crosses x days Shan-i-Punjab x Prabhat 5.79 a Trt x crosses x days * Values with the same letters are not significantly different according to Fisher s LSD test at 5% level (2.00mg/L) + IBA (1.00 mg/l)]. Embryo germination in M 3 and M 4 medium (75.52 and %) was not differed significantly. In embryo rescue at different growth stages, all the hybrids showed highest embryo germination in M 2 medium and lowest in M 6 medium. In embryo rescue of 65 days aged embryos, Shan-i-Punjab x Florda Prince showed highest germination (75.23 %), whereas, in embryo rescue of 75 and 85 days aged embryo Shan-i-Punjab x Prabhat recorded maximum germination (84 % and % respectively) in the same medium i.e M 2 and minimum germination was found in Shani-Punjab x Flordaglo cultured in M 5 medium in all growth stages. When growth regulators were used at lower concentration maximum embryo germination was recorded as compared to the increased level of growth regulators. This is in accordance with the findings of Liu et al., (2007) who studied different media and concentrations of hormones for successful embryo culture and suggested that the abortion of embryo occured due to improper hormone balance whereas excess hormones induced the formation of callus (Fuller et al., 2011). Data further reveals that cultivars and growth stages had significant effect on embryo germination. Among all crosses Shan-i-Punjab x Prabhat recorded mean highest germination (77.69 %) followed by Shan-i-Punjab x Florda Prince (74.50 %) and minimum in Shan-i-Punjab x Flordaglo (71.97 %). These results of different hybrids response to embryo germination are confirmatory with the results of Hamill et al., (2005) who found a significant differences in cultivar response to germination. Among different growth stages, maximum germination (78.05 %) was recorded when embryos were rescued at full maturity (85 days after pollination) followed by 75 days after pollination (75.14 %) and minimum germination (70.97 %) in 65 days old embryo. Similarly, Theobold and Hough (1960) reported significant differences in terms of embryo growth in immature and fully ripen fruits of peach. Infante and Gonzalez (2002) also recorded maximum percentage of embryo germination when embryos were harvested from the most advanced mature peach fruits. Our results of determining the best embryo age for in vitro germination is contradictory to the results of Srivastav et al., (2004) who reported that 60 days aged embryos of Saharanpur Prabhat and Flordasun, either open-pollinated or crossed with each other showed better culture establishment, maturation and germination than 70 and 80 days aged embryos. Data pertaining to the effect of embryo age and WP medium on shoot length of low chill peach hybrids is given in Table 2. Mean longest shoot was recorded in M 3 [WP + BAP (0.50mg/ L) + IBA (0.10mg/L)] and M 4 medium [WP + BAP (1.00mg/L) + IBA (0.25 mg/l)] which was 5.90 and 5.77 cm respectively. Plants with shortest shoot (4.95 cm) was recorded in M 6 media [WP + BAP (2.00mg/L) + IBA (1.00 mg/l)]. Different growth stages affected the shoot length significantly. Mean maximum shoot length (5.94 cm) was observed in plants rescued from 85 days aged embryos, followed by embryos rescued after 75 days of pollination (5.55 cm) and mean minimum shoot length (5.08 cm) was found in embryos cultured 65 days after pollination. In embryos harvested after 65 and 85 days of pollination, Shan-i-Punjab x Florda Prince recorded longest shoots (5.31cm and 6.25 cm respectively) whereas, in embryo 501

4 INDIRA DEVI et al., Table 3: Response of embryo age and WP media supplemented with growth regulators on root length of low chill peach hybrids Treatments 65 DAP 75 DAP 85 DAP MS + Growth Regulators (mg/l) Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Shan-i-Punjab Shan-i-Punjab Shan-i-Punjab Mean Mean x x x x x x x x x Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat Florda Prince Flordaglo Prabhat M 0 : Control b* 1 M : BAP (0.125) + IBA (0.01) b 2 M : BAP (0.25)+ IBA (0.05) b 3 M : BAP (0.50)+ IBA (0.10) b 4 M : BAP (1.00)+ IBA (0.25) a 5 M : BAP (1.50)+ IBA (0.50) c 6 M : BAP (2.00)+ IBA (1.00) c Mean Days mean 5.57 b 5.76 b 6.85 a Crosses Mean Shan-i-Punjab Florda 5.93 b LSD 0.05 x Prince : Trt Crosses 0.19 Days 0.19 Trt x crosses Trt x days Shan-i-Punjab x Flordaglo 5.78 b Crosses x days Shan-i-Punjab x Prabhat 6.47 a Trt x crosses x days * Values with the same letters are not significantly different according to Fisher s LSD test at 5% level rescue of 75 days old embryo, Shan-i-Punjab x Prabhat recorded maximum shoot length (5.69 cm). Shan-i-Punjab x Florda Prince and Shan-i-Punjab x Prabhat did not differ significantly for shoot length. In Shan-i-Punjab x Prabhat longest shoot (5.79 cm) was recorded and shortest in Shan-i-Punjab x Flordaglo (5.28 cm). Similar trend of shoot length was observed in all crosses with minimum shoot length in those embryos harvested after 65 days of pollination and maximum in those which were harvested 85 days after pollination. Theobold and Hough (1960) also reported significant differences in terms of embryo growth in immature and fully ripen fruits of peach. For root length also embryo age showed significant effect (Table 3). Among different concentrations of growth regulators, embryos raised in M 4 medium [WP + BAP (1.00mg/L) + IBA (0.25 mg/l)] recorded mean longest roots (6.76 cm). This media recorded longest roots in all hybrids rescued at different growth stages, whereas embryos grown in M 0, M 1, M 2 and M 3 medium didn t show significant effect on root length. Similarly in M 5 and M 6 medium non significant differences in root length were recorded. In three growth stages of embryo culture, embryo rescued after 85 days of pollination showed better results with mean maximum root length of 6.85 cm whereas embryos harvested after 65 and 75 days of pollination showed non significant differences for root length (5.76 cm and 5.57 cm respectively). All crosses showed increased root length with increasing the embryo age. Among crosses, mean longest roots were recorded in Shan-i- Punjab x Prabhat (6.47 cm). Shan-i-Punjab x Florda Prince and Shan-i-Punjab x Flordaglo did not show significant difference for root length. After 65, 75 and 85 days of pollination, maximum root length was recorded in Shan-i- Punjab x Prabhat (5.76 cm, 5.98 cm and 7.70 cm respectively) and minimum in Shan-i-Punjab x Flordaglo (5.43 cm and 5.40 cm respectively) but in 85 days aged embryos, minimum root length was recorded in Shan-i-Punjab x Florda Prince (6.36 cm). This difference in root length might be because of the genotypic differences, which is in accordance with the findings of Cornu et al., (1981) who reported the effect of genotype on roots of cultured embryos. REFERENCES Anderson, N. and Byrne, D. H Cooler temperate during germination improves germination and survival of embryo cultured peach seed. Acta. Hort. 592: Bacon, T. A. and Byrne, D. H Per cent dry weight of the ovule predicts peach seed viability. Hort. Sci. 40: Centallas, Q. A., Raseira, M., Silva, J. B., Silva, J. B., Citadin, I., Silveria, C. A. P. and Da-silva, J. B Germination and development of seeds of early ripening peach using embryos culture. Agropecuaria-Clima-Temperado. 3: Cornu, D., Riffaud, J. L. and Capelli, P In vitro propagation of wild cherry tree (Prunus avium L.). Proc IUFRO sect. S Int Workshop in vitro for tree species. Fontainebleau, France., pp Fathi, H., Arzani, K., Ebadi, A. and Khalighi, A Production of hybrid sweet cherry plants (silej delamarka and zard daneshkadeh) with embryo culture method. Seed Plt. Imp. J. 25: Fuller, R. J., Liddiard, V. M., Hess, J. R. and Carman, J. G Improving cotton embryo culture by simulating in ovulonutrient and 502

5 EMBRYO RESCUE OF LOW CHILL PEACH HYBRIDS AS AFFECTED hormone levels. Vitro. Cellular. Develop. Bio-Plant. 47: Hamill, S. D., Beppu, K., Topp, B. L., Russell, D. M. and DeFaveri, J Effects of media and fruit ripeness on germination and transplanting of in vitro cultured embryos from low chill peach and nectarine. Acta. Hort. 694: Infante, R. and Gonzalez, J Early maturing peach embryo rescue and in vitro survival at different fruit growth stages. Acta. Hort. 592: Jeengool, N. and Boonprakob, D Rescue of peach embryo in culture media with additional of 6-benzyladenine and gibberellic acid. Nat. Sci. 38: Kumar, J., Thakur, A., Verma, M. K., Kumar, K., Singh, H., Singh, D., Thakur, D.,Jawandha, S. K. and Banyal, S. K Peach. In: Temperate Fruit Crop Breeding: domestication to cultivar development Part II (eds. S. N. Ghosh, M. K. Verma and A. Thakur), Jaya Publishing, New Delhi. pp Liu, W., Chen, X., Liu, G., Liang, Q., He, T. and Feng, J Interspecific hybridization of Prunus persica with P. armeniaca and P. salicina using embryo rescue. Plant. Cell. Tiss. Organ. Cult. 88: Mancuso, M. L., Caruso, T. and Germana, M. A Peach breeding programme for early ripening, low chilling requirement cultivars: embryo rescue and somatic embryogenesis. Acta. Hort Perez - Hernandez, J. B. and Grajal-Martin, M. J In vitro culture of immature zygotic embryos mango and plantlet development. Hort. Sci. 46: Srivastav, M. Bal, K. and Arora, R. L Determination of embryo age for in vitro culture establishment and enhanced germinability in early maturing subtropical peach cultivars. Indian. J. Hortic. 61: Theobald, W. L. and Hough, L. F The relationship between stages of peach embryo development and seedling growth survival. Proc. Am. Soc. Hort. Sci. 75: Uma, S., Lakshmi, S., Saraswathi, M. S., Akbar, A. and Mustaffa, M. M Embryo rescue and plant regeneration in banana (Musa spp). Plant Cell Tiss Org Cult 105:

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