Production of Hybrid Rice seeds using environment sensitive genic male sterile (EGMS) and basmati rice lines in Kenya

Photoperiod-sensitive genic male sterile rice (PGMS) lines IR-73827-23-76-15-7 S, IR-75589-31-27-8-33S referred to as P1 and P2, and IR-77271-42-25-4-36S, thermo-sensitive genic male sterile (TGMS) line referred to as T were obtained from International Rice research Institute. These lines, collectively known as environment genic male sterile lines, were sown under greenhouse growth conditions where temperatures were more than 34°C with an objective of inducing complete male gamete sterility in them. Results indicated that high temperature growth conditions induces complete male gamete sterility in both the PGMS and TGMS lines. The impact of this is that, it will be possible to produce pure basmati hybrid rice seed in the tropical regions without contamination with pure breed lines. The male sterile PGMS/TGMS were pollinated with pollen from basmati370 and 217 grown under natural conditions and some hybrid seeds were obtained. This shows that high temperature emasculated the male gametes but not female ones. The conclusion is that it is possible to induce complete male gamete sterility in PGMS and TGMS under greenhouse in tropical growth conditions, and to produce hybrid rice seeds. This makes basmati hybrid rice seed production in Kenya a viable venture.


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The World rice production was about 503.6million tons in 2017 (1). This is below consumption that 38 was 505.8 million tons in the same period. In Kenya, rice consumption is over 580,000 tonnes against 39 a total production of about 149,000 tonnes (2). The deficit, which is valued at over Kenya shillings 40 Seven billion is imported (2). Basmati rice is preferred by many consumers compared to non-aroma 41 varieties because of its good cooking traits (3). However, in Kenya, basmati yields only 3.6 to 42 4.0tones per hectare (4). This is quite low and it has contributed to keeping its prices high. Over the 43 years, rice breeding has gone through a breeding paradigms with emphasis of high yield (HYV) 44 semi-dwarf varieties (5). The major shift came with the green revolution which brought about IR8 45 variety in 1966 with the dwarf gene sd-1 (6) which raised the yield to over 6 tones per hectare (7). 46 Hybrid rice technology was introduced in 1970s (8, 9) to improve yield above dwarf lines. Heterosis 47 improved yields in rice (10, and hybrids lines are reported to have a 20-25 percent yield advantage 48 over pure breeds (11). However, some advantages of this have been eroded by diseases such as blast 49 (12). To overcome this, green super hybrid technology has been adopted that further increased 50 realizable rice yield per hectare by 12% above the normal hybrids (13). Advances in green super 51 hybrid technology started in China in 1996 and it targeted raising rice grain yield from about 10 52 tones to about 17 tones per hectare (14). The yield was realizable by combining hybrid vigour and 53 good agronomic traits such as disease resistance (15). According to Yuan Longping (16), rice yield 54 in China stands at about 17 tones per hectare. 55 A number of approaches have been used in hybrid rice production that include the three line system, 56 which utilizes cytoplasmic male sterility (CMS) (17) and the two line hybrid system that are referred 6 57 to as environment sensitive genic male sterility (EGMS) (18)(19)(20). Among the EGMS is the 58 photoperiod-sensitive genic male sterile (PGMS) rice line that is completely sterile when under 59 14hours daylight length growth conditions. It reverts to fertility in varying degree when grown under 60 less than 14 hours daylight length conditions (18,19). Other EGMS are thermosensitive genic male 61 sterile (TGMS) rice lines that is sterile when grown under high temperature and revert to some 62 fertility when grown under low temperature growth conditions (17). In their sterile phase the EGMS 63 rice lines are crossed with a male parent to produce F 1 (hybrid) seeds (21).

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Pure basmati rice yield per hectare is low compared to non-aromatic lines (22) and this has kept its 65 prices high. Basmati370 and 217 varieties are the two major aromatic rice varieties grown in Kenya.

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Exploitation of hybrid technology to improve their yield is limited (23). Elsewhere, attempts to 67 produce hybrid basmati rice lines have shown a good combining abilities in yield traits (24). In this 68 research object was to produce basmati370 and 217 hybrid rice seeds using two line methods. The 69 yields traits realized were better than that of both purebred lines.  plates in a nursery until seedlings were 21 days old. Transplanting of seedlings in the field was done 85 at spacing of 20cm x 20cm in growth troughs made of concrete blocks in the greenhouse (GH).

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Control seedlings were sown outside the greenhouse under natural growth conditions. In each set  (CRP) which is 30 days before heading, EGMS and basmati parents were exposed to high 115 temperature under GH growth conditions till heading when female plants were pollinated with 116 pollen from male parents. Glumes were clipped at the tips to expose the stigma then pollen from 117 fertile basmat370 and B217 was dusted over the clipped glumes between 11.30pm and 1.30pm.

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Pollinated panicles were then bagged to prevent unwanted crossings.

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Hybrids and parental lines were planted in a complete randomised block design (3 blocks with 3 121 replicates). All standard agronomic practises such as pest and diseases control were done. Yield and a seed counter used to get 1000 seeds that was used to determine grain weight. Days to heading 126 were determined at 50% emergence of panicles starting from the sowing date, while days to maturity 127 was calculated as 30 plus days to 50% heading of each rice line. The percentage seed set rate was 128 determined using equation below;

Data analysis
132 Data obtained on temperature, parental pollen viability, height, productive tillers, flowering date, 133 seed setting, panicle length and exertion ANOVA was analysed using SPSS 16.0 statistical package.

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Numerical data of two environments was expressed in Mean±SD and analysed using studentst-test 135 for significance. At p≤0.05, mean values, were considered statistically significant.

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The temperatures in the greenhouse (GH) and outside greenhouse (OGH) growth conditions were 139 by average 24˚c and 34˚c respectively. Within GH growth conditions, line P1, T and P2 recorded 140 pollen fertility of less than 2% while basmati370 and 217 recorded 25% and 21% respectively. All 141 lines grown under OGH conditions recorded over 60% pollen fertility (Fig 1). The results effectiveness of GH to raise temperature and effectively induce complete sterility in 158 EGMS and subjected to unpaired T-test analysis are shown in table 1. Line P1 with 2.4*10 -11 had the 159 highest pollen sterility rate compared to basmati370 with5.6*10 -12 sterility levels when grown uder 160 GH coditions. On the other hand P1 with 6.9*10 -11 had lowest fertility levels compared to basmati370 161 that had 1.1*10 -4 (highest) among the parents under OGH growth conditions. However, there was 162 no significance difference in pollen sterility under GH and OGH growth conditions among all the 163 parental lines (Table 1a). Some F1 seeds obtained in each cross breed are as recorded in Table 2 164 11 165 Over 80% of anthers locules from EGMS grown outside the greenhouse conditions, were filled with 168 conspicuous pollen grains (Fig 2a), but locules for EGMS grown under greenhouse growth 169 conditions had no observable pollen grains (Fig 2b). The EGMS grown OGH and inside GH had 170 their staining yellow and spikelets had no observable grains (Figs 2a and b However, most pollen 171 for EGMS OGH stained blue black and spites were conspicuous filled with grain (Figs 3 c and d).      (Table 4). with 1% potassium iodide (Figs 1 and 2). This is an indication that their pollen were completely Greenhouse induced day-time temperature of above 34 o C was able to completely induce male 254 sterility among P1, P2 and T with over 98% sterility (Tables 2-3). Many of the pollen were of 255 abortive type and it stained yellow with 1% potassium iodide. EGMS exposed to high temperature 256 had as low as less 2% seed set rate. It means use of staining method is accurate method of monitoring 257 spikelet fertility. The EGMS exposed to temperature of around 24 o C under natural environment seed set rate than EGMS (Fig 1). This is an indication that they do not have thermo/photo sensitive 264 male sterility genes like the EGMS. Therefore, they can be used as pollen donor in hybrid rice 265 production programme.

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Pollen sterility in lines P1, P2 and T grown under GH was over 97% and with a seed set rate of less 268 2% (Table 4) p≤0.05for days to heading ( Table 5). The EGMS varieties P1 had the highest p-value followed by 281 P2 (Table 1). Also, sterility is influenced by the level of temperature which influences the overall 282 level of pollen viability (Fig 1). This explains why lines P1, T, and P2 did not have seeds under GH  Lines TB217 and TB370 were better than the rest in anthesis (AD), days to heading (HD), and days 287 to maturity (MD) (   and 12hours of light length). This will allow production of basmati rice seeds in Kenya, using EGMS.

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Yield traits, such as grain weight showed better performance in hybrid than the best performing The EGMS can be tested areas of Kenya hotter than Mwea to test ability to produce hybrids outside 351 greenhouse growth conditions.