The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.)

Transcription

1 Plant Cell Rep (2002) 20: DOI /s CELL BIOLOGY AND MORPHOGENESIS M. Y. Zheng Y. Weng W. Liu C. F. Konzak The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.) Received: 12 April 2001 / Revised: 22 October 2001 / Accepted: 23 October 2001 / Published online: 14 December 2001 Springer-Verlag 2001 Abstract Microspores were isolated from wheat (Triticum aestivum L.) spikes of various genotypes following an effective pretreatment that induced microspore embryogenesis. The isolated microspores were cultured with or without live ovaries, and with or without medium pre-conditioned by ovaries for varying periods of time. Live ovaries alone increased androgenic embryoid yields up to 4.5-fold over the control for microspores isolated from responsive genotypes. While live ovary co-culture alone was not effective for microspores isolated from recalcitrant genotypes, the addition of medium preconditioned by ovaries to microspore cultures increased embryoid yield by more than 100-fold. Without ovary-conditioned medium, no embryoids could be obtained from some recalcitrant genotypes. Ovary-conditioned medium apparently functions to increase embryoid yields by providing essential substance(s) for elaboration of the embryogenic program already triggered during pretreatment. Keywords Embryogenesis Embryoid Microspore culture Nurse factors Ovary conditioning Wheat Abbreviations DH: Doubled haploid NPB: Northwest Plant Breeding Co OVCM: Ovary-conditioned medium Introduction Communicated by J.M. Widholm M.Y. Zheng ( ) Y. Weng W. Liu C.F. Konzak Northwest Plant Breeding Co, 2001 Country Club Rd, Pullman, WA 99163, USA mzheng63@yahoo.com Tel.: , Fax: Even though embryogenic microspores may naturally occur in the anthers of many plant species (Guha and Maheshwari 1964; Chuang et al. 1978; Heberle-Bors 1985; Raghavan 1986; Ziauddin et al. 1990), the frequencies are usually low. An efficient system for the production of doubled haploids through microspore embryogenesis requires the artificial manipulation of immature pollen and subsequent success in plant regeneration (Hu et al. 1995; Touraev et al. 1996a, b; Hu and Kasha 1997; Touraev et al. 1997; Liu et al. 2001; Zheng et al. 2001). Such manipulations often consist of two critical steps, the first of which involves attempts to switch from the naturally determined pollen-forming developmental program of microspores to the inherent alternative developmental program that leads to the induction of their embryogenic competency (Reynolds 1997; Zheng et al. 2001). The second step is to provide an adequate environment for induced microspores to elaborate their induced embryogenic program. Favorable factors during this phase may include both optimized physical conditions such as temperature and osmolarity and proper ingredients in the media, including carbohydrate, plant hormones, mineral nutrients, vitamins, etc. (Mejza et al. 1993; Tuvesson and Öhlund 1993; Gustafson et al. 1995). If conditions are sub-optimal, many competent microspores may either fail to divide or terminate their divisions, hence abort their embryogenic process, which, in effect, substantially reduces the yields of microsporederived plants (Zhou et al. 1991; Ziauddin et al. 1992; Zheng and Konzak 1999; Zheng et al. 2001). One aspect that has been found to be critical for microspore embryogenesis is the co-cultivation of isolated microspores with live ovaries (Bruins et al. 1996; Puolimatka et al. 1996; Hu and Kasha 1997). When growing ovaries were co-cultured with microspores, androgenic embryoid yields were observed to increase and green plant regeneration also improved. The inclusion of ovaries in these culture systems seemed to enhance embryogenesis and/or improve the quality of the embryoids. The genotypes tested in these studies, however, were all responsive to culture procedures that did not use live ovaries. In the investigation reported here, we studied the effect and role of live ovaries as well as culture media preconditioned by ovaries on microspore embryogenesis by both responsive and non-responsive (or recalcitrant) genotypes. There were three objectives: (1) to test

2 the effect of the addition of medium preconditioned by ovaries; (2) to find the optimal time period and density for ovary preconditioning; (3) to determine if the proper use of live ovaries and/or ovary-conditioned medium would enhance DH production in wheat (Triticum aestivum L.), especially in recalcitrant genotypes. Materials and methods Growing donor plants Spring wheat cultivars Chris, Pavon 76, WED (characterized as responsive to culture) as well as Waldron (a North Dakota public cultivar) and WPB 926 (owned by Western Plant Breeders), both recalcitrant genotypes, were grown in a greenhouse. Two seeds were sown in each pot (20 25 cm). Plants were grown at 27±2 C/16±2 C under a 17/7-h (light/dark) photoperiod. Fertilizers (20:20:20; N:P:K) were pre-mixed with soil at the time the seed were sown. Further applications of fertilizer were achieved by daily watering with water containing liquid forms of nitrogen, phosphorus and potassium at a ratio of 20:20:20 plus trace or micronutrient minerals. Morphological features of the tillers containing microspores at mid- to late uninucleate stage were established for each genotype via microscopic examination of microspores stained with acetocarmine. Tillers that contained microspores at the mid- to late uninucleate stage were cut with two nodes remaining from the top and immediately placed in a 250-ml flask with distilled water. All leaf blades were removed by cutting at their bases. Fresh tillers so collected were then pretreated (Liu et al. 2001). Preparation and use of OVCM and live ovaries There were two types of co-culture involved in this research. One was the co-culture of microspores with only live ovaries at the time of culture initiation. The other was the co-culture of microspores with OVCM. Co-culture of microspores with OVCM in all experiments was a two-step process. The first step dealt with the production of OVCM by which live ovaries were excised to condition medium NPB-99 (Zheng et al. 2001). Live ovaries were isolated aseptically from disinfected spikes using a pair of fine forceps and placed into petri dishes (15 60 mm), each containing 5 ml of NPB-99 medium. These petri dishes were then sealed and incubated at 27 C for conditioning. Ovary densities and conditioning periods varied with experiments. The next step (once the conditioning is completed) was the dilution of OVCM with regular NPB-99 for use in microspore cultures. An aliquot of 1.0 ml OVCM was drawn from each of the conditioned plates and mixed with regular NPB-99 to a final volume of 4 ml for subsequent use for microspore culture. For some experiments, live ovaries at a concentration of two per 1 ml of regular medium were picked and placed into petri dishes to co-culture with the microspores. In the last experiment, both live ovaries and OVCM, alone or in combination, were also added to microspore cultures. Except when otherwise noted, all of the ovaries used were obtained from spikes of Chris, an awnless cultivar. Microspore cultures and experiments Microspores were isolated and purified following the procedures described by Liu et al. (2001). They were then divided equally into various plates to a final density of microspores per milliliter of culture medium. Microspore density was assessed using a haemocytometer and an inverted microscope. Once the OVCM was mixed with regular medium and isolated microspores, all petri dishes were sealed and incubated in the dark at 27 C for embryoid induction. In all experiments, there were four plates (hence four 803 replicates; n=4) for each particular treatment. For counting induced cells, dividing cells and proembryoids, four spots in each petri dish were randomly selected and scored under an inverted microscope. A hand-held numerical device was used for the actual counting. All frequencies were calculated based upon the total number of microspores. For treatments from which the numbers of embryoids were too great to carry out actual counting, numbers in embryoids above 500 were derived from counting the first 500 embryoids and then estimating the total on the basis of the unit weight of the first 500. The first group of experiments was designed to test if one dose of OVCM would enhance the embryogenic development of microspores leading to increases in embryoid yields. OVCM was prepared by fixing the ovary density at ten per milliliter of medium. The conditioning periods varied from 7 days to 40 days, during which time 1 ml OVCM was withdrawn at 7, 14, 21, 28 and 40 days and mixed with 3 ml NPB-99 medium for microspore culture. Data on the frequencies of initial embryogenic microspores and the percentages of dividing cells, proembryoids and embryoids were collected. All data presented in the tables are means ± standard errors. Observations on the pace of androgenic embryoid development were also made at various times during the culture using the inverted microscope. The second group of experiments was designed to estimate the most effective conditioning period and the optimum density of ovaries to be used for preparation of the OVCM. First, a fixed ovary density of ten ovaries per milliliter of medium was used to condition media for 4, 7, 10, 14, 21, 28, 35, 37 and 40 days in an effort to find the best conditioning period. Once the most effective conditioning period was identified, OVCM from conditioning by various densities of ovaries at 2, 5, 10, 15, 20, and 30/ml but withdrawn on the same day for microspore cultures were compared for their effectiveness in microspore embryogenesis. The third group of experiments was carried out to compare the effectiveness of optimized OVCM with or without live ovary coculture with five representative genotypes Chris, Pavon 76, Waldron, WED and WPB 926. The best treatment factors from the second group of experiments were used. A co-culture of 0.5 ml OVCM (from 10 days of conditioning by ten ovaries per milliliter of medium) and five live ovaries with microspores in 4 ml medium in petri dishes was also tested. Microspore cultures with neither live ovaries nor OVCM were included as blank controls. Results and discussion The effects of a single dose of OVCM on microspore embryogenesis Compared with the control, a single dose of OVCM that had been conditioned by ovaries for 7 28 days enhanced microspore embryogenesis, as indicated by the higher percentage of proembryoids and higher embryoid yields per spike from these microspore cultures (Table 1). OVCM that had been conditioned for 37 days or 40 days could not sustain cell divisions by the majority of microspores toward mature embryoids and, consequently, led to a lower number of embryoids per spike. The results showed that a prolonged conditioning over 28 days produced OVCM that was negative with respect to embryoid yields when compared to the control (Table 1). For microspores cocultured with live ovaries but no OVCM, embryoids were still developing at the time of counting, while all of the embryoids from cultures with OVCM had reached the size for transfer to regeneration medium by 28 days from culture initiation. This seemed to suggest that OVCM accel-

3 804 Table 1 The effectiveness of ovary co-culture and ovary preconditioned medium mixed with regular medium at a 1:3 ratio in maintaining embryogenesis of microspores isolated from cv. Chris Control Live ovary Conditioning period (days) Induced total percentage a 31±2.7 41±0.9 20±2.5 19±3.1 19±1.8 22±1.2 29±0.9 40±2.7 Cell division (%) b 21±3.1 37±4.1 10±1.7 11±0.7 9±0.9 18±2.3 19±0.8 30±3.2 Percentage proembryoids 1.9± ± ± ± ± ± ± ±3.2 Number of embryoids per spike 127±34 5,820±315 87±21 117±34 946±187 1,140±279 2,768±243 4,506±408 a Each number is a mean (n=4) ± standard error b Data on percentage of cell division were collected 8 days following culture initiation Table 2 Effect of the length of the conditioning period of OVCM by ovaries on the embryogenesis of microspores a Control Conditioning period (days) Live ovary Cell division 25±4.4 20±2.8 19±3.2 17±3.0 19±3.7 22±0.7 34±2.6 47±4.2 50±6.4 49±7.7 41±1.1 (%) b Percentage 1.7± ± ± ± ± ±1.4 16± ± ± ± ±2.8 proembryoids Percentage 0.5± ± ± ± ± ± ± ± ± ± ±1.6 embryoids a Microspores were isolated from Pavon 76 using the flask system (Liu et al. 2001), while ovaries were obtained from genotype Bob White. The density for conditioning was ten ovaries per milliliter. One milliliter OVCM was mixed with 3 ml regular NPB-99 medium for microspore culture b Data on percentage of cell division were collected 3 days following culture initiation erated androgenic embryoid development in microspores compared to those cultured with only live ovaries. Such an acceleration might be attributed to the immediate availability of nurse factors (Xu et al. 1981) that had accumulated in the OVCM during the conditioning. However, the total number of embryoids that developed from microspores co-cultured with live ovaries was significantly higher than that from microspores cultured with a single dose of OVCM (Table 1). This trend also held true for other responsive genotypes. Taken together, these results suggest that although one dose of OVCM provides enough nurse factors to initiate and sustain embryogenesis, a sustained release of nurse factors by the continuous presence of ovaries in the induction medium exerted even greater benefits. As the ovaries grew and expanded in size in the culture, they continued to release more nurse factors for up to 4 weeks (data not shown), thereby enabling the production of more embryoids. Such results are in agreement with earlier studies from the same or different responsive genotypes in wheat microspore cultures (Bruins et al. 1996; Puolimatka et al. 1996; Hu and Kasha 1997). Even though the addition of OVCM appeared to accelerate cell divisions and microspore embryogenesis, it may require more than a single dose of OVCM to maximize the embryogenic potential of all cultured microspores. The optimum density and the best conditioning period by ovaries OVCM that had been conditioned by ten ovaries per milliliter for various time periods were compared for their effectiveness in stimulating microspore embryogenesis. OVCM that had been conditioned for 7 10 days produced the highest percentage of embryoids when compared to OVCM that had been conditioned for any of the other time periods (Table 2), showing that the positive effects of OVCM peaked around 7 10 days of conditioning. An effective OVCM accelerated microspore cell divisions, thereby resulting in an earlier and more synchronized production of mature embryoids. In cultures with OVCM, the first batch of embryoids reached the size that they could be transferred for plant regeneration after 21 days, 1 week earlier than the controls, which started with live ovaries but without OVCM in the microspore culture medium. Again, the final values in both the percentage of proembryoids and percentage of embryoids from microspores co-cultured with live ovaries were higher than those from microspores co-cultured with a single dose of OVCM. In an experiment designed to find the optimum ovary density for conditioning, microspores were isolated from cv. Pavon 76. When 1 ml OVCM that had been conditioned for 10 days by 2, 5, 10, 15, 20 and 30 ovaries per milliliter, respectively, was used for microspore culture, OVCM from conditioning with five ovaries per milliliter or less was observed to be sub-optimal as embryoid

4 805 Table 3 The effectiveness of a 10-day conditioning by ovaries at various densities on microspore embryogenesis a Control Live ovary Density of ovaries per milliliter for conditioning Percentage cell division b 29.4± ± ± ± ± ± ± ±2.7 Percentage proembryoids 1.8± ± ± ± ± ± ± ±3.2 Number of embryoids per spike 137±47 5,264± ±153 1,474±310 4,369±385 4,052±310 3,986±632 3,810±175 a Microspores were isolated from Chris. One milliliter OVCM was mixed with 3 ml regular NPB-99 medium for microspore culture b Data on percentage of cell division were collected 3 days following the culture initiation Table 4 Effect of the presence of live ovaries, OVCM or a combination of the two on embryoid yields per spike from various genotypes in microspore cultures Treatment Responsive genotypes Recalcitrant genotypes Chris Pavon 76 WED 202 Waldron WPB 926 Control (no additions) 126±29 147±18 86±15 3±2 0±0 10 live ovaries 5,962±407 6,387±393 3,794±307 2±2 0±0 1 ml OVCM a 3,759±538 4,732±405 2,968± ±93 678± ml OVCM+5 live ovaries 5,783±348 5,969±336 3,814± ±69 823±107 a 0.5 ml or 1 ml OVCM was mixed with regular NPB-99 medium to a final volume of 4 ml for microspore cultures yields were substantially lower than all other treatments except the control. One milliliter OVCM that had been conditioned by 10 or 15 ovaries per milliliter was the most beneficial for microspore embryogenesis, as shown by the higher percentage of dividing cells, higher percentage of proembryoids and higher number of embryoids per spike (Table 3). A further increase in ovary density to 20 or 30 ovaries per milliliter for conditioning led to reduced embryoid yields compared to these obtained in OVCM conditioned with 10 or 15 ovaries per milliliter (Table 3). The cause of these decreases in embryoid yields is not clear. It could be due to an excessive consumption of nutrients in the OVCM by excess ovaries or to the toxicity of some unknown factor. In subsequent experiments, 1 ml OVCM that had been conditioned for 10 days by ten ovaries per milliliter was further diluted into 4 ml by regular medium; when used, it was found to be effective for the 25 wheat genotypes tested thus far, including Bob White, Chris, PV76, Waldron, WED and WPB 926. The effects of OVCM combined with live ovary co-culture on microspore embryogenesis by recalcitrant genotypes When microspores isolated from the recalcitrant genotypes Waldron and WPB 926 were cultured with live ovaries or no ovaries at all, a very low number of mature embryoids, or no mature embryoids at all, were produced. Microspore cultures with OVCM or a combination of OVCM and live ovaries produced a significantly higher embryoid yield (Table 4). For Waldron and WPB 926, microspore cultures with OVCM alone produced 471 and 678 embryoids, respectively, while those with a 0.5 ml OVCM + five live ovaries as co-culture yielded 595 and 823 embryoids, respectively, representing over a 100-fold increase over the control (Table 4). When the same combination was applied to microspores from the responsive genotypes Chris, Pavon 76 and WED , no significant differences were observed between co-culture with ten live ovaries and coculture with 0.5 ml OVCM + five live ovaries. The number of embryoids that developed in the 1 ml OVCM coculture was lower than that in the 0.5 ml OVCM + 5 five live ovary combination, and no co-culture or co-culture with only live ovaries again produced the lowest number of embryoids (Table 4). This clearly supports the concept that nutrients or nurse factors released by the ovaries are stimulatory to microspore embryogenesis. Even for the responsive genotypes, the total absence of ovaries in culture resulted in a substantially lower yield of embryoids, as has also been reported by others (Bruins et al 1996; Puolimatka et al. 1996; Hu et al 1997). The dramatic effect of OVCM appears to result from its ability to accelerate microspore divisions. The positive response shown by microspores of recalcitrant genotypes to the combination of OVCM and live ovary coculture indicates that the addition of OVCM at an early stage in culture was essential for boosting the embryogenesis of these microspores. This might mean that within a very narrow time window during the early stages of the embryogenic development by the microspores of recalcitrant genotypes, a functionally minimal level of essential substance(s) or nursing factors acted to stimulate efficient cell divisions of the reprogrammed microspores, thereby leading to embryoid formation. Once this time window has passed, the loss of embryogenic potential by the microspores of the recalcitrant genotypes appears to be irreversible, because adding OVCM to mi-

5 806 crospore cultures of these genotypes after 7 days did not help the embryogenic process nor prevent the degeneration of microspores (data not shown). Microscopic examination of microspores from recalcitrant genotypes also showed that the first cell divisions in cultures with OVCM started by 18 h, while no divisions were observed until 36 h or later in cultures without OVCM. Moreover, without OVCM, the frequency of dividing microspores was lower and cell divisions began to halt approximately 7 10 days from culture initiation, leaving no potential for mature embryoid formation. The fact that the most effective OVCM was that conditioned between 7 days and 10 days indicates that the nurse factors reached a peak level during that period. Considering that microspore division in recalcitrant genotypes began to stop at approximately the same time period when without OVCM, it is reasonable to propose that a boosting dose of nurse factors, acquired through the addition of OVCM, seemed to be crucial (may be essential) at the time the microspores started dividing and certainly before the halt began (7 10 days in culture). This may also help to explain why live ovary co-culture alone was ineffective in promoting microspore embryogenesis in recalcitrant genotypes the slow release of nurse factors by live ovaries was insufficient to trigger the microspores during the brief receptive period. By the time enough nurse factors were released by these ovaries, cell divisions had already halted, making it impossible to produce mature embryoids. A one-time infusion of nurse factors through OVCM at the culture initiation helped the microspores to achieve optimal divisions and development towards embryoids. On the other hand, cell divisions by microspores from responsive genotypes started normally by 24 h in culture, even without ovary co-culture or the addition of OVCM. The time window that permits nurse factors to take effect in responsive genotypes may be wider. In other words, microspores from responsive genotypes might have a much higher tolerance to a temporary lack of nurse factors. This in turn gave live ovaries time to grow and release enough nurse factors to facilitate a continued embryogenic development towards embryoids. Since the combination of OVCM and live ovary coculture appears to be essential for recalcitrant genotypes and not detrimental to responsive genotypes, it is advisable to use such combination in all microspore cultures, especially for samples whose culture responsiveness is unknown. Preliminary experiments showed that OVCM can be stored at 4 C for up to 10 days but that its effectiveness declines with time (data not shown). To ensure an adequate supply of nurse factors, aged ovaries in co-culture may be replaced with fresh ones after 28 days. For a better understanding of the role of OVCM or ovary coculture, one must first identify and characterize the functional components released into the medium by ovaries. Nevertheless, the use of OVCM has helped to produce large numbers of DH plants, even from the recalcitrant genotypes. Work is now in the planning stage for characterizing the active nurse factors released by wheat ovaries. Genotype differences in the effectiveness of the ovaries used have not been found so far. OVCMs produced by ovaries from a wide range of genotypes, also ranging from the early uninucleate through to the binucleate stages, were all effective in promoting microspore embryogenesis. The density of ovaries used and the length of the conditioning period seemed to be more critical than the genotype from which the ovaries were obtained. Conditioning with ten ovaries per milliliter of medium for a period of 7 10 days and a subsequent dilution of 1 ml OVCM with 3 ml liquid medium in induction culture was the most beneficial with respect to embryoid development. In conclusion, the addition of live ovaries to culture media is beneficial for microspore embryogenesis of responsive genotypes. More importantly, the employment of OVCM, alone or in combination with live ovary coculture, is a key to effectively obtaining mature embryoids from genotypes previously considered to be recalcitrant. Using this strategy, we produced large numbers of embryoids from numerous genotypes that were otherwise non-responsive to procedures either with or without live ovary co-culture. Future efforts should be devoted to characterizing such nurse factors that are released through in vitro culture of live ovaries. 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