Growing and Pollinating Maize   

Materials and Reagents

1. 1.5 ml centrifuge tubes
   
2. Soil for large pots (Multipurpose Blend)
   
3. Soil for trays and peet pots (Supersoil^® Potting Soil)
   
4. Shoot bags: ‘Lawson 217’ 5000 bags/box
   
5. Tassel bags: ‘Lawson 402’ 1000 bags/box
   Note: Colored bags are useful in a field with few pollinations.
   
6. Slow release fertilizer (Osmocote 14N-14P-14K; A90550)
   
7. Gnatrol (for insect control, available from Valent; EPA Reg. No. 73049-56)
   Note: It must be used in compliance with Worker Protection Standard, 40 CFR Part 170.
   
8. Field stakes (Hummert International)
   
9. Row cover (Agribon)
   
10. Staples
    
11. Paperclips
    

Equipment

1. Planter (Hand Jab Slim-Style Planter)
   
2. 11.35 L (3-gallon) pots (such as 3 gal pot from Viagrow available at Amazon.com)
   
3. Planting Trays (such as F1721 from T.O. Plastics available at Hummert International)
   
4. Plastic mulch for the field and drip system (Hummert International)
   
5. Drying oven set to 38 °C (such as those manufactured by Despatch)
   
6. Walk-in cooler with low humidity (such as those manufactured by Norlake)
   

Procedure

1. Planting
   1. Control environment
      1. Plant kernels directly into 11.35 L pots or into peat pots for later transplanting. Cover kernels with 2-3 cm of soil and pre-water with Gnatrol at a concentration of 15 ml to 7.5 L water. We use Supersoil for trays and peat pots and a blend of soil for the 11.35 L pots (see ‘Materials and Reagents’).
         Note: If transplanting, the ideal time is at V2, when the third leaf is just visible. Any later, the male and female flowers may not be in synchrony, making self-pollinations difficult. Planting the same material twice with a one-week gap provides the possibility of crossing siblings plants if self-pollinations can’t be performed.
         
      2. Make a hole in the soil and insert the peat pot containing the seedling. Water thoroughly when finished transplanting.
         Note: The pots receive 15 ml of slow release fertilizer (14:14:14 NPK) at the time of transplanting.
         
      3. The plants are ideally fertilized 3 times a week through a drip system (20:20:20 NPK). If no drip system is available, hand-water with a hose and continue using slow release fertilizer.
         
   2. Field environment
      1. The ‘Hand Jab Slim Style Planter’ is useful for precision field planting. A spacing of 20 cm between plants within a row is sufficient unless the plants make lots of tillers. A combination of black plastic mulch with drip tape is ideal. The seeds should be close (~5 cm) to the drip tape. An alternative to the black plastic mulch is hand-weeding. Watering can also be accomplished by flooding an adjacent furrow or via the rain. Weeding is essential for corn, especially at the early stages.
         
      2. Fertilize at seedling emergence and two weeks later. Fertilizer recommendations include a mixture of nitrogen, potassium and phosphorus. Amounts are highly dependent on the soil and age of the plant. Details can be found at: http://apps.cdfa.ca.gov/frep/docs/Corn.html.
         
      3. Provide a sturdy, well-labeled stake at the front of each family. Plan for the possibility of stakes being knocked down by keeping a map and maintaining a gap between families.
         
      4. Pests vary by location but crows are ubiquitous and may pull up seedlings, so consider covering the planted seeds with row cover. Remove the row cover at 3-4 weeks.
      
      
2. Pollination
   1. Preparing ears for pollinations
      Cover ears with shoot bags prior to silk emergence (Figure 1D). Usually, ears are ready for shoot-bagging when the tassel first appears (condition known as protogyny), which is 50-90 days after planting, depending on the genotype. In some genotypes, silks are ‘sneaky’ and emerge before the husk leaves are visible. For critical plants or when there are limited inbreds, shoot-bag the top two ears. At the peak of the field season, especially in windy fields, plants need to be checked daily as shoot bags blow off or ears burst through the bags.
      
   2. Preparing tassels for pollination
      1. Pollen can be taken from a male tassel as soon as anthers dehisce (Figure 1A). The tassel starts shedding on the main rachis, then the branches. Usually a tassel will shed for 5-7 days unless it is very hot or has no branches in which it may shed out in 1-2 days.
         
      2. Bag the tassel with a ‘Lawson 402’ bag before pollinations; this waiting period ensures that the live pollen comes from the bagged tassel and is not undesired pollen that blew onto the tassel from a neighbor. Any contaminating pollen will be dead after 12 h and the fresh pollen gathered will be from the bagged plant.
         
      3. Label the bag with the respective female/male and date of pollination. Conventionally, the female parent is written first. The label must be wax coated and the details must be written with pencil or permanent marker. Holding the bag in one hand, slide the tassel in with the other. The bag does not need to be completely opened up to slide onto the tassel. For videos on maize pollinations, go to https://www.maizegdb.org/controlled_pollination.
         
      4. There are many ways to fold the bag (Figures 1B and 1C) but the important point is that the bag tightly grabs the tassel rachis but is not so tight that the tassel breaks. It can be stapled or held with a paperclip.
         
   3. Cutting back silks
      The ear is ready when silks have developed. In some genotypes, the silks are visible through the translucent shoot bag, in others, it is a matter of feeling the ear for plumpness. In either case, pinch the cob gently and make a cut about 5 cm above the tip of the cob and the cut silks should be visible. A pocket knife or scissors can be used. The cut surface of silks is not receptive to pollen since pollen germinates into the side of the silks. After cutting, replace the shoot bag and wait until the next day. The silks will have grown 1-2 cm overnight (Figure 1E). This compact cluster of silks makes pollinations more controlled.
      
      
      Figure 1. Pollinating maize. A. Tassel with anthers that are shedding pollen. B-C. Two ways to fold a tassel bag. The bag is stapled below the flag leaf which is visible in the fold of the bag. D. A plant with shoot bag on the ear. E. Shoot bag removed to show the cut silks, ready for pollination.
      
      
   4. Pollination
      1. Having bagged the tassel and cut back the silks the day before, gently bend the male plant over and tap on the bag holding the tassel and open the staples. This tapping releases the pollen from the anthers. Slide the bag off the tassel and open it up to see the pollen. If many anthers have fallen into the bag, you can tap these out without losing your pollen. There will be enough pollen for many females, but 3-4 is a realistic number. Be careful if it is windy–minimize the amount of time that the bag is open to avoid pollen contamination. Similarly, avoid the condition where pollen remains on your hands after successive pollinations from different genotypes.
         
      2. Remove the shoot bag from the female and tap the pollen onto the silks. Quickly cover the ear with the labeled tassel bag and staple it around the ear and culm.
   5. Harvest
      1. Approximately 30 days after pollination, the water can be turned off from the field. The kernels are ready for harvest about 45 days from pollination.
         
      2. Dry the ears in a hot air oven at 35-40 °C or less for 3-7 days until kernels easily come off the cob. If there is no oven, the ears can be left on a dry table in the greenhouse for one to two weeks.
         
      3. Ideally, the kernels are stored in a cool room (10 °C and low humidity (30%)). With such conditions, the kernels remain viable for 20 years.

Acknowledgments

CL is supported by NSF IOS-1238202 and SH by USDA CRIS 5335-21000-013-00D. The authors declare no conflicts of interest.

References

1. Candela, H. and Hake, S. (2008). The art and design of genetic screens: maize. Nat Rev Genet 9(3): 192-203.